Rapid Evaporative Ionisation Mass Spectrometry (&#34;REIMS&#34;) and Desorption Electrospray Ionisation Mass Spectrometry (&#34;DESI-MS&#34;) Analysis of Swabs and Biopsy Samples

ABSTRACT

A method is disclosed comprising providing a biological sample on a swab, directing a spray of charged droplets onto a surface of the swab in order to generate a plurality of analyte ions, and analysing the analyte ions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/033,447, filed on Jul. 12, 2018, which is a continuation of U.S.patent application Ser. No. 15/555,783, filed on Sep. 5, 2017, now U.S.Pat. No. 10,026,599, which is a U.S. National Phase of InternationalApplication No. PCT/GB2016/050621 entitled “Rapid Evaporative IonisationMass Spectrometry (“REIMS”) and Desorption Electrospray Ionisation MassSpectrometry (“DESI-MS”) Analysis of Swabs and Biopsy Samples” filedMar. 7, 2016, which claims priority from and the benefit of UnitedKingdom patent application No. 1503876.3 filed on Mar. 6, 2015, UnitedKingdom patent application No. 1503864.9 filed on Mar. 6, 2015, UnitedKingdom patent application No. 1518369.2 filed on Oct. 16, 2015, UnitedKingdom patent application No. 1503877.1 filed on Mar. 6, 2015, UnitedKingdom patent application No. 1503867.2 filed on Mar. 6, 2015, UnitedKingdom patent application No. 1503863.1 filed on Mar. 6, 2015, UnitedKingdom patent application No. 1503878.9 filed on Mar. 6, 2015, UnitedKingdom patent application No. 1503879.7 filed on Mar. 6, 2015 andUnited Kingdom patent application No. 1516003.9 filed on Sep. 9, 2015.The entire contents of these applications are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates generally to mass spectrometers and inparticular to the analysis of material by ambient ionisation ion sourcesincluding rapid evaporative ionisation mass spectrometry (“REIMS”) ionsources and the analysis of material by desorption electrosprayionisation (“DESI”) mass spectrometry. Various embodiments relate to theuse of mass spectrometers in diagnostic methods. Various embodiments arecontemplated wherein analyte ions generated by an ambient ionisation ionsource are then subjected either to: (i) mass analysis by a massanalyser such as a quadrupole mass analyser or a Time of Flight massanalyser; (ii) ion mobility analysis (IMS) and/or differential ionmobility analysis (DMA) and/or Field Asymmetric Ion MobilitySpectrometry (FAIMS) analysis; and/or (iii) a combination of firstly ionmobility analysis (IMS) and/or differential ion mobility analysis (DMA)and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysisfollowed by secondly mass analysis by a mass analyser such as aquadrupole mass analyser or a Time of Flight mass analyser (or viceversa). Various embodiments also relate to an ion mobility spectrometerand/or mass analyser and a method of ion mobility spectrometry and/ormethod of mass analysis.

BACKGROUND

The mucosal membrane is a protective layer responsible for trappingpathogens in the human body. The mucosal membrane is an easilyaccessible and highly clinically relevant sample to diagnose pathogenicand cancerous associated diseases.

It is known to use medical swabs as a standard collection device formucosal membranes.

Conventionally, the swab is placed into a sterile tube containing abuffer solution for storage and then the tube is sent to a laboratoryfor analysis. A laboratory receiving the tube will wipe the smearcontent across a culture medium such as an agar plate. The culturemedium is then incubated to allow organisms present on the swab to grow.

Microbial identification may then be performed, e.g., under amicroscope. Any organisms present in the sample may also be identifiedby 16S gene-sequencing and/or by using matrix-assisted laser desorptionionisation (“MALDI”) mass spectrometry and then comparing the massspectra with a commercial available database.

Although easy to handle, the current approach to the analysis of medicalswabs for diagnostic purposes is culture-dependent and involves a timeconsuming and costly workflow. Diagnosis of diseases, such as infectionsor dysbiosis, and appropriate treatment is therefore associated withconsiderable delay. Furthermore, around 95% of bacteria cannot becultured for analysis.

It is therefore desired to provide an improved method for mucosalanalysis, e.g., diagnosis.

SUMMARY

According to an aspect there is provided a method comprising:

providing a sample on a swab;

directing a spray of electrically charged droplets onto a surface of theswab in order to generate a plurality of analyte ions; and

analysing the analyte ions.

Desorption electrospray ionisation (“DESI”) is an ambient ionisationmethod that involves directing a spray of (primary) electrically chargeddroplets onto a surface. The electrospray mist is pneumatically directedat the sample where subsequent splashed (secondary) droplets carrydesorbed ionised analytes. After ionisation, the resulting ions travelthrough air and pass into an atmospheric pressure interface of a massspectrometer. Desorption electrospray ionisation (“DESI”) is a techniquethat allows for ambient ionisation of a trace sample at atmosphericpressure with no sample preparation being required.

A “swab” in accordance with various embodiments is intended to beunderstood as comprising a “standard medical swab” i.e. a swab that isdesigned for sampling biological samples such as mucosal membranes. Forexample, the term “standard medical swab” should be understood ascovering a “cotton bud” (British) or a “cotton swab” (American) i.e. asmall wad of cotton wrapped around one or both ends of a tube. The tubemay be made from plastic, rolled paper or wood.

StableFlex® fibre cores are known and comprise a 80 μm fused silica corecoated with a polymer. Such fibre cores are not intended to beconsidered as comprising a “swab” within the meaning of the presentapplication as materials having a fibre core are not considered tocomprise a “standard medical swab”.

In particular, the terms “swab”, “medical swab” and “standard medicalswab” as used within the present application are intended to excludematerials having a fused silica core.

It is known to directly analyse Solid Phase Micro Extraction (“SPME”)fibres using desorption electrospray ionisation (“DESI”) massspectrometry. In this technique, a SPME fibre is exposed to the headspace within a vial containing a sample. Alternatively, a SPME fibre maybe dipped into a sample in solution. According to the known technique,analyte may be extracted into a SPME fibre coating in the liquid or gasphase, i.e. such that the sample does not remain in its native state.According to the known technique, the SPME fibre is then subjected todirect desorption electrospray ionisation (“DESI”) mass spectrometryanalysis by positioning the fibre in the spray of a desorptionelectrospray ionisation (“DESI”) mass spectrometry arrangement.

Accordingly, this known technique requires (at least) a two-step samplepreparation procedure comprising: (i) the acquisition or preparation ofthe sample, followed by (ii) the extraction of analyte into the SPMEfibre. Moreover, the known specialist SPME fibres are not suited for thedirect sampling of biological material, e.g. from a patient, such as thedirect sampling of the mucosal membrane.

In contrast, various embodiments are based at least in part upon therecognition that a sample provided on a swab, e.g. a standard medicalswab, may be directly analysed by desorption electrospray ionisation(“DESI”) mass spectrometry. In particular, an important aspect ofvarious embodiments is that a standard medical swab may itself bepositioned in the spray of a desorption electrospray ionisation (“DESI”)ionisation source.

Various embodiments are beneficial in that they require no samplepreparation steps beyond the acquisition of the sample onto the swab.

Accordingly, various embodiments provide a rapid direct analysis methodfor samples provided on medical swabs.

Various embodiments are particularly suited to and useful in theanalysis of biological material, e.g. from a patient, such as materialsampled directly onto a swab from the mucosal membrane.

Furthermore, the use of medical swabs in desorption electrosprayionisation (“DESI”) mass spectrometry analysis according to variousembodiments opens up the possibility of performing multiple differentanalyses of the same sample. For example, multiple different analysescan be performed on the same swab. Such an approach beneficiallyprovides multiple sets of information or data related to the same samplein a particularly convenient and efficient manner.

This ability to perform multiple different analyses on the same swab isdue to the fact that desorption electrospray ionisation (“DESI”) massspectrometry analysis is a relatively non-destructive analysistechnique. Furthermore, other commercial analysis techniques, such asculturing techniques and 16S rRNA sequencing techniques are optimised touse samples provided on (standard) medical swabs.

Accordingly, following a single sample acquisition onto a swab, thesample on the swab may be analysed multiple times using multipledifferent analysis techniques.

It will be appreciated, therefore, that various embodiments provideimproved methods for analysis, e.g., diagnosis.

The terms “biological sample”, “biological material” and the like areused interchangeably herein unless otherwise specified and they mayoptionally comprise or consist of biological tissue. The biologicalmaterial etc. may optionally be selected, for example, from a surgicalresection specimen, a biopsy specimen, a tissue specimen, a cellspecimen, a smear, a body fluid specimen and/or a faecal specimen, anyof which may be sampled in vivo from the subject, e.g., directly sampledonto a swab or taken as a biopsy, or be sampled ex vivo or in vitro,e.g., a sample may be provided and then sampled onto a swab, which maybe referred to as indirect sampling onto a swab, or sampled with abiopsy needle, which may be referred to as indirect sampling with abiopsy needle. Optionally, the method is carried out on a providedbiological sample, e.g., a provided biopsy, or a provided swab, i.e. aswab onto which a biological material was previously sampled.

A body fluid specimen may, for example, optionally be selected fromblood, plasma, serum, sputum, lavage fluid, pus, urine, saliva, phlegm,vomit, faeces, amniotic fluid, cerebrospinal fluid, pleural fluid,semen, sputum, vaginal secretion, interstitial fluid, and/or lymph.

The swab may comprise a medical swab or a standard medical swab.

The swab may comprise a disposable swab.

The swab may comprise a cotton, rayon, plastic or foam swab.

The swab may comprise a hollow rod.

The swab may comprise plastic, wood or rolled paper.

The swab may comprise a swab arranged and adapted to sample a mucosalmembrane.

The swab may have been chemically modified to enhance selectivity for ananalyte.

The chemical modification may render the swab lipophilic.

The chemical modification may involve formation of a coating on asurface of the swab.

The coating may be a polymeric coating.

The polymer coating may comprise polydivinylbenzene (DVB), a copolymerof N-vinylpyrrolidone and divinylbenzene, or polydimethylsiloxane.

The chemical modification may utilise a solid-phase extraction material.

The polymeric coating may contain particles of a solid-phase extractionmaterial.

The solid-phase extraction material may comprise polymer particles,silica particles, hybrid silica/organic particles, carbon particles orpolymer-coated solid particles.

The polymer-coated solid particle may be a silica particle.

The polymer-coated solid particles may comprise polydivinylbenzene(DVB), a copolymer of N-vinylpyrrolidone and divinylbenzene, orpolydimethylsiloxane.

The silica particles may be surface modified by reaction with a surfacemodifier having the formula:

Z_(a)(R′)_(b)Si—R²,

wherein Z═Cl, Br, I, C₁-C₅ alkoxy, dialkylamino ortrifluoromethanesulfonate; a and b are each an integer from 0 to 3provided that a+b=3; R¹ is a C₁-C₆ straight, cyclic or branched alkylgroup, and R² is a functionalizing group.

R′ may be selected from the group consisting of methyl, ethyl, propyl,isopropyl, butyl, t-butyl, sec-butyl, pentyl, isopentyl, hexyl andcyclohexyl.

R² may include an alkyl, alkaryl, alkenyl, alkynyl, aryl, cyano, amino,diol, nitro, ester, a cation exchange group, an anion exchange group, analkyl or aryl group containing an embedded polar functionality or chiralmoieties.

R² may be C₁-C₃₀ alkyl, alkaryl, cyanoalkyl, a diol; alkylamino, aminoalkyl or carbamate group.

The silica particles may be surface modified by reaction with a compoundselected from the group consisting of octyltrichlorosilane,octadecyltrichlorosilane, octyldimethylchlorosilane andoctadecyldimethylchlorosilane.

The polymer particles may comprise polydivinylbenzene or a copolymer ofN-vinylpyrrolidone and divinylbenzene.

The solid-phase extraction material may be an ion exchange resin.

The polymer particles may be ion exchange resin particles.

The ion exchange resin may comprise a copolymer of N-vinylpyrrolidoneand divinylbenzene in which at least some of the benzene rings aresulfonated or carboxylated.

The ion exchange resin may comprise a copolymer of N-vinylpyrrolidoneand divinylbenzene in which at least some of the benzene rings aresubstituted by imidazolium, —CH₂-piperazine groups or quaternaryammonium groups.

The quaternary ammonium group may be —CH₂N⁺(CH₃)₂(C₄H₉).

The solid-phase extraction material or solid particle may be attached tothe swab using an adhesive.

The sample may comprise biological tissue, biological matter, abacterial colony, a fungal colony, and/or microbes.

The sample may be provided on the swab in its native or unmodifiedstate.

The sample may comprise: (i) mammalian cells; (ii) microbes; (iii)extracellular or exogenous compounds; and/or (iv) a biomarker of (i),(ii) and/or (iii).

The biomarker may be selected from fatty acids, glycerolipids, sterollipids, sphingolipids, prenol lipids, saccharolipids, and/orphospholipids, and/or a fingerprint of one or more thereof.

Providing the sample on the swab may comprise wiping the swab across anin vivo, in vitro or ex vivo biological sample or more generally atarget. According to an embodiment the target may comprise an organicsample including a plastic. The target may comprise one or morebacterial colonies or one or more fungal colonies.

Providing the sample on the swab may comprise using the swab to sample amucosal membrane.

The mucosal membrane may comprise a vaginal, nasal or oral mucosalmembrane.

Directing the spray of charged droplets onto the swab may comprisedirecting a spray of charged solvent droplets onto the swab.

Directing the spray of charged droplets onto the swab may comprisedirecting the spray of charged droplets onto the swab at aboutatmospheric pressure.

Directing the spray of charged droplets onto the swab may compriseionising the sample using Desorption Electrospray Ionisation (“DESI”) ordesorption electroflow focusing ionisation (“DEFFI”).

The method may comprise directing the spray of charged droplets onto theswab from a sprayer.

The sprayer may be arranged at a distance from the swab in the range (i)<0.5 mm; (ii) about 0.5-1 mm; (iii) about 1-1.5 mm; (iv) about 1.5-2 mm;(v) about 2-3 mm; (vi) about 3-4 mm; or (vii) >4 mm.

The method may comprise providing the sprayer with a voltage in therange (i) <1 kV; (ii) about 1-2 kV; (iii) about 2-3 kV; (iv) about 3-4kV; (v) about 4-5 kV; (vi) about 5-6 kV; (vii) about 6-7 kV; (viii)about 7-8 kV; (ix) about 8-9 kV; (x) about 9-10 kV; or (xi) >10 kV.

The method may comprise providing the sprayer with a solvent at a flowrate in the range (i) <5 μL/min; (ii) about 5-7 μL/min; (iii) about 7-9μL/min; (iv) about 9-10 μL/min; (v) about 10-11 μL/min; (vi) about 11-13μL/min; (vii) about 13-15 μL/min; (viii) about 15-20 μL/min; or (ix) >20μL/min.

The method may comprise providing the sprayer with a gas at a pressurein the range (i) <5 bar; (ii) about 5-6 bar; (iii) about 6-7 bar; (iv)about 7-8 bar; (v) about 8-9 bar; (vi) about 9-10 bar; (vii) about 10-15bar; or (viii) >15 bar.

The gas may comprise air or nitrogen.

The method may comprise rotating the swab whilst directing the spray ofcharged droplets onto the surface of the swab.

The step of rotating the swab whilst directing the spray of chargeddroplets onto the surface of the swab may comprise substantiallycontinuously rotating the swab whilst directing the spray of chargeddroplets onto the surface of the swab.

The method may comprise translating and/or oscillating the swab whilstdirecting the spray of charged droplets onto the surface of the swab.

The step of translating and/or oscillating the swab may comprisesubstantially continuously translating and/or oscillating the swabwhilst directing the spray of charged droplets onto the surface of theswab.

The method may comprise translating and/or oscillating the swabsubstantially in the direction of the axial length of the swab.

Analysing the analyte ions may comprise transferring the analyte ions toa mass and/or ion mobility spectrometer via a capillary or other inlet.

The entrance to the capillary or other inlet may be arranged at adistance from the swab in the range (i) <0.5 mm; (ii) about 0.5-1 mm;(iii) about 1-1.5 mm; (iv) about 1.5-2 mm; (v) about 2-3 mm; (vi) about3-4 mm; or (vii) >4 mm.

Analysing the analyte ions may comprise mass analysing and/or ionmobility analysing the analyte ions or ions derived from the analyteions to obtain mass spectrometric data and/or ion mobility data.

Various embodiments are contemplated wherein analyte ions generated byan ambient ionisation ion source are then subjected either to: (i) massanalysis by a mass analyser or filter such as a quadrupole mass analyseror a Time of Flight mass analyser; (ii) ion mobility analysis (IMS)and/or differential ion mobility analysis (DMA) and/or Field AsymmetricIon Mobility Spectrometry (FAIMS) analysis; and/or (iii) a combinationof firstly ion mobility analysis (IMS) and/or differential ion mobilityanalysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS)analysis followed by secondly mass analysis by a mass analyser or filtersuch as a quadrupole mass analyser or a Time of Flight mass analyser (orvice versa). Various embodiments also relate to an ion mobilityspectrometer and/or mass analyser and a method of ion mobilityspectrometry and/or method of mass analysis.

Analysing the analyte ions may comprise determining the ion mobility,collision cross section or interaction cross section of the analyte ionsor ions derived from the analyte ions to obtain mass spectrometric dataand/or ion mobility data.

The method may comprise analysing the mass spectrometric data and/or ionmobility data in order either: (i) to distinguish between healthy anddiseased states; (ii) to distinguish between potentially cancerous andnon-cancerous states; (iii) to distinguish between different types orgrades of cancer; (iv) to distinguish between different types or classesof sample; (v) to determine whether or not one or more desired orundesired substances are present in the sample; (vi) to confirm theidentity or authenticity of the sample; (vii) to determine whether ornot one or more impurities, illegal substances or undesired substancesare present in the sample; (viii) to determine whether a human or animalpatient is at an increased risk of suffering an adverse outcome; (ix) tomake or assist in the making a diagnosis or prognosis; (x) to inform asurgeon, nurse, medic or robot of a medical, surgical or diagnosticoutcome; and/or (xi) to identify and/or predict one or more diseases orclinical disorders.

The one or more diseases or clinical disorders may comprise (i) aninfection; (ii) a change in the microbiome; (iii) pre-term delivery inpregnancy; (iv) an immunological disorder; (v) asthma; (vi) an allergy;(vii) inflammation; (viii) cancer; (ix) necrosis; and/or (x) apre-cancerous state.

The method may comprise determining whether cancerous biological tissueor the tumour comprises either: (i) grade I, grade II, grade III orgrade IV cancerous tissue; (ii) metastatic cancerous tissue; (iii) mixedgrade cancerous tissue; or (iv) a sub-grade cancerous tissue.

The step of analysing the mass spectrometric data may compriseperforming a supervised and/or unsupervised analysis of the massspectrometric data.

The step of analysing the mass spectrometric data and/or ion mobilitydata may comprise using one or more of: univariate analysis;multivariate analysis; principal component analysis (PCA); lineardiscriminant analysis (LDA); maximum margin criteria (MMC);library-based analysis; soft independent modelling of class analogy(SIMCA); factor analysis (FA); recursive partitioning (decision trees);random forests; independent component analysis (ICA); partial leastsquares discriminant analysis (PLS-DA); orthogonal (partial leastsquares) projections to latent structures (OPLS); OPLS discriminantanalysis (OPLS-DA); support vector machines (SVM); (artificial) neuralnetworks; multilayer perceptron; radial basis function (RBF) networks;Bayesian analysis; cluster analysis; a kernelized method; and subspacediscriminant analysis.

The method may comprise analysing the sample on the swab using one ormore further different analysis methods.

The one or more further different analysis methods may comprise aculturing analysis method.

The culturing analysis method may comprise wiping the swab across aculturing medium, incubating the culturing medium, and examining theculturing medium under a microscope.

The one or more further different analysis methods may comprise a genesequencing method.

The gene sequencing method may comprise a 16S rRNA gene sequencingmethod.

The one or more further different analysis methods may comprise aMatrix-Assisted Laser Desorption Ionisation (“MALDI”) method.

The one or more further different analysis methods may comprise anambient ionisation mass spectrometry method.

The one or more further different analysis methods may comprise a RapidEvaporative Ionisation Mass Spectrometry (“REIMS”) method.

According to an aspect there is provided apparatus comprising:

a first device arranged and adapted to direct a spray of chargeddroplets onto a surface of a swab in order to generate a plurality ofanalyte ions; and

a second device arranged and adapted to analyse the analyte ions.

The first device may be arranged and adapted to direct a spray ofcharged solvent droplets onto the swab.

The first device may be arranged and to direct the spray of chargeddroplets onto the swab at about atmospheric pressure.

The first device may comprise a Desorption Electrospray Ionisation(“DESI”) ion source or a desorption electroflow focusing ionisation(“DEFFI”) ion source.

The first device may comprise a sprayer arranged and adapted to directthe spray of charged droplets onto the swab.

The sprayer may be arranged at a distance from the swab in the range (i)<0.5 mm; (ii) about 0.5-1 mm; (iii) about 1-1.5 mm; (iv) about 1.5-2 mm;(v) about 2-3 mm; (vi) about 3-4 mm; or (vii) >4 mm.

The apparatus may comprise a device arranged and adapted to provide thesprayer with a voltage in the range (i) <1 kV; (ii) about 1-2 kV; (iii)about 2-3 kV; (iv) about 3-4 kV; (v) about 4-5 kV; (vi) about 5-6 kV;(vii) about 6-7 kV; (viii) about 7-8 kV; (ix) about 8-9 kV; (x) about9-10 kV; or (xi) >10 kV.

The apparatus may comprise a device arranged and adapted to provide thesprayer with a solvent at a flow rate in the range (i) <5 μL/min; (ii)about 5-7 μL/min; (iii) about 7-9 μL/min; (iv) about 9-10 μL/min; (v)about 10-11 μL/min; (vi) about 11-13 μL/min; (vii) about 13-15 μL/min;(viii) about 15-20 μL/min; or (ix) >20 μL/min. The apparatus maycomprise a device arranged and adapted to provide the sprayer with a gasat a pressure in the range (i) <5 bar; (ii) about 5-6 bar; (iii) about6-7 bar; (iv) about 7-8 bar; (v) about 8-9 bar; (vi) about 9-10 bar;(vii) about 10-15 bar; or (viii) >15 bar.

The gas may comprise air or nitrogen.

The apparatus may comprise a third device arranged and adapted to rotatethe swab whilst the spray of charged droplets is directed onto thesurface of the swab.

The third device may be arranged and adapted to substantiallycontinuously rotate the swab whilst the spray of charged droplets isdirected onto the surface of the swab.

The apparatus may comprise a fourth device arranged and adapted totranslate and/or oscillate the swab whilst the spray of charged dropletsis directed onto the surface of the swab.

The fourth device may be arranged and adapted to substantiallycontinuously translate and/or oscillate the swab whilst directing thespray of charged droplets onto the surface of the swab.

The fourth device may be arranged and adapted to translate and/oroscillate the swab substantially in the direction of the axial length ofthe swab.

The apparatus may comprise a capillary or other inlet arranged andadapted to transfer the analyte ions to the second device.

The entrance to the capillary or other inlet may be arranged at adistance from the swab in the range (i) <0.5 mm; (ii) about 0.5-1 mm;(iii) about 1-1.5 mm; (iv) about 1.5-2 mm; (v) about 2-3 mm; (vi) about3-4 mm; or (vii) >4 mm.

The second device may comprise a mass analyser or filter and/or ionmobility analyser arranged and adapted to mass analyse and/or ionmobility analyse the analyte ions or ions derived from the analyte ions.

The second device may comprise an ion mobility device arranged andadapted to determine the ion mobility, collision cross section orinteraction cross section of the analyte ions or ions derived from theanalyte ions.

According to an aspect there is provided a medical swab for use in amethod as described above, wherein the swab has been chemically modifiedto enhance selectivity for an analyte.

The swab may be a disposable swab.

The swab may be a cotton, rayon, plastic or foam swab.

The chemical modification may render the swab lipophilic.

The chemical modification may involve formation of a coating on asurface of the swab.

The coating may be a polymeric coating.

The polymer coating may comprise polydivinylbenzene (DVB), a copolymerof N-vinylpyrrolidone and divinylbenzene, or polydimethylsiloxane.

The chemical modification may utilise a solid-phase extraction material.

The polymeric coating may contain particles of a solid-phase extractionmaterial.

The solid-phase extraction material may comprise polymer particles,silica particles, hybrid silica/organic particles, carbon particles orpolymer-coated solid particles.

The polymer-coated solid particle may be a silica particle.

The polymer-coated solid particles may comprise polydivinylbenzene(DVB), a copolymer of N-vinylpyrrolidone and divinylbenzene, orpolydimethylsiloxane.

The silica particles may be surface modified by reaction with a surfacemodifier having the formula:

Z_(a)(R′)_(b)Si—R²,

wherein Z=C1, Br, I, C₁-C₅ alkoxy, dialkylamino ortrifluoromethanesulfonate; a and b are each an integer from 0 to 3provided that a+b=3; le is a C₁-C₆ straight, cyclic or branched alkylgroup, and R² is a functionalizing group.

R′ may be selected from the group consisting of methyl, ethyl, propyl,isopropyl, butyl, t-butyl, sec-butyl, pentyl, isopentyl, hexyl andcyclohexyl.

R² may include an alkyl, alkaryl, alkenyl, alkynyl, aryl, cyano, amino,diol, nitro, ester, a cation exchange group, an anion exchange group, analkyl or aryl group containing an embedded polar functionality or chiralmoieties.

R² may be C₁-C₃₀ alkyl, alkaryl, cyanoalkyl, a diol; alkylamino, aminoalkyl or carbamate group.

The silica particles may be surface modified by reaction with a compoundselected from the group consisting of octyltrichlorosilane,octadecyltrichlorosilane, octyldimethylchlorosilane andoctadecyldimethylchlorosilane.

The polymer particles may comprise polydivinylbenzene or a copolymer ofN-vinylpyrrolidone and divinylbenzene.

The solid-phase extraction material may be an ion exchange resin.

The polymer particles may be ion exchange resin particles.

The ion exchange resin may comprise a copolymer of N-vinylpyrrolidoneand divinylbenzene in which at least some of the benzene rings aresulfonated or carboxylated.

The ion exchange resin may comprise a copolymer of N-vinylpyrrolidoneand divinylbenzene in which at least some of the benzene rings aresubstituted by imidazolium, —CH₂-piperazine groups or quaternaryammonium groups.

The quaternary ammonium group may be —CH₂N⁺(CH₃)₂(C₄H₉).

The solid-phase extraction material or solid particle may be attached tothe swab using an adhesive.

According to an aspect there is provided a method of chemicallymodifying a medical swab which comprises attaching particles of asolid-phase extraction material to the surface of the swab.

The attachment may utilise an adhesive.

According to an aspect there is provided a method of chemicallymodifying a medical swab comprising:

(a) forming a dispersion or solution of a chemical modifier;

(b) dipping the swab into the solution or dispersion;

(c) removing the swab from the solution or dispersion and drying it.

Steps (b) and (c) may be repeated at least once.

According to an aspect, there is provided a diagnostic methodcomprising:

providing a sample on a swab wherein the swab further may comprise asolid-phase extraction (“SPE”) substance for extracting analyte from aliquid sample;

directing a spray of electrically charged droplets onto a surface of theswab in order to generate a plurality of analyte ions; and

mass analysing the analyte ions.

The solid-phase extraction substance may comprise a polymer.

The solid-phase extraction substance may be hydrophilic.

The solid-phase extraction substance may comprise a reversed-phasepolymer.

The solid-phase extraction substance may comprise a cation-exchange oranion-exchange polymer.

The solid-phase extraction substance may comprise octadecylsilane(“ODS/C18”) or polydimethylsiloxane (“PDMS”).

The solid-phase extraction substance may comprise: (i) Oasis MAX(mixed-mode cation exchange)®; (ii) hydrophilic N-vinylpyrrolidone andlipophilic divinylbenzene or Oasis HLB(hydrophilic-lipophilic-balanced)®; or (iii) Oasis MCX (mixed-modecation exchange)®.

The method may further comprise rotating the swab whilst directing thespray of electrically charged droplets onto the surface of the swab.

The step of rotating the swab whilst directing the spray of electricallycharged droplets onto the surface of the swab further may comprisesubstantially continuously rotating the swab whilst directing the sprayof electrically charged droplets onto the surface of the swab.

The swab may comprise cotton, rayon or polyester.

The swab may comprise fibres which do not have a fused silica core.

According to an aspect there is provided a diagnostic method comprising:

providing a sample on a swab wherein the swab further may comprise asolid-phase extraction (“SPE”) substance for extracting analyte from aliquid sample;

rotating the swab and at substantially the same time directing a sprayof electrically charged droplets onto a surface of the swab in order togenerate a plurality of analyte ions; and

mass analysing and/or ion mobility analysising the analyte ions.

According to an aspect there is provided a method of DesorptionElectrospray Ionisation (“DESI”) comprising a diagnostic method asdescribed above.

According to an aspect there is provided a method of mass spectrometryand/or method of ion mobility analysis comprising a method as describedabove.

According to an aspect there is provided apparatus comprising:

a device for directing a spray of electrically charged droplets onto asurface of a swab in order to generate a plurality of analyte ions,wherein the swab further may comprise a solid-phase extraction (“SPE”)substance for extracting analyte from a liquid sample; and

a mass analyser or filter and/or ion mobility analyser for massanalysing and/or ion mobility analysing the analyte ions.

According to an aspect there is provided apparatus comprising:

a first device for directing a spray of electrically charged dropletsonto a surface of a swab in order to generate a plurality of analyteions, wherein the swab further may comprise a solid-phase extraction(“SPE”) substance for extracting analyte from a liquid sample;

a second device for rotating the swab at substantially the same time asthe first device directs the spray of electrically charged droplets ontothe surface of the swab; and

a mass analyser or filter and/or ion mobility analyser for massanalysing and/or ion mobility analysing the analyte ions.

According to an aspect there is provided a mass and/or ion mobilityanalyser comprising apparatus as described above.

According to an aspect there is provided a method comprising:

providing a sample on a swab;

wetting the swab with a first liquid;

contacting the wetted swab with an electrode in order to generate anaerosol, smoke or vapour;

generating a plurality of analyte ions from the aerosol, smoke orvapour; and

analysing the analyte ions.

According to an aspect there is provided a diagnostic method comprising:

providing a sample on a swab;

wetting the swab with a first liquid;

contacting the wetted swab with a bipolar electrode in order to generatean aerosol;

generating a plurality of analyte ions from the aerosol; and

mass analysing and/or ion mobility analysing the analyte ions.

The first liquid may comprise water.

According to an aspect there is provided a method of Rapid EvaporativeIonisation Mass Spectrometry (“REIMS”) comprising a method as describedabove.

According to an aspect there is provided apparatus comprising:

an electrode which is arranged and adapted to contact a sample on a swabwhich has been wetted with a first liquid so as to generate an aerosol,smoke or vapour;

a device which is arranged and adapted to generate a plurality ofanalyte ions from the aerosol, smoke or vapour; and

an analyser for analysing the analyte ions.

According to an aspect there is provided apparatus comprising:

a bipolar electrode which is arranged and adapted to contact a sample ona swab which has been wetted with a first liquid so as to generate anaerosol;

a device which is arranged and adapted to generate a plurality ofanalyte ions from the aerosol; and

a mass analyser or filter and/or ion mobility analyser for massanalysing and/or ion mobility analysing the analyte ions.

According to an aspect there is provided a mass and/or ion mobilityspectrometer comprising apparatus as described above.

According to an aspect there is provided a swab comprising a solid-phaseextraction (“SPE”) substance for extracting analyte from a liquidsample, wherein the swab comprises fibres which do not have a fusedsilica core.

The swab may comprise cotton, rayon or polyester.

The solid-phase extraction substance may be hydrophilic.

The solid-phase extraction substance may comprise a reversed-phasepolymer.

The solid-phase extraction substance may comprise a cation-exchange oranion-exchange polymer.

The solid-phase extraction substance may comprise octadecylsilane(“ODS/C18”) or polydimethylsiloxane (“PDMS”).

The solid-phase extraction substance may comprise: (i) Oasis MAX(mixed-mode cation exchange)®; (ii) hydrophilic N-vinylpyrrolidone andlipophilic divinylbenzene or Oasis HLB(hydrophilic-lipophilic-balanced)®; or (iii) Oasis MCX (mixed-modecation exchange)®.

According to an aspect there is provided a method comprising:

providing a sample on a swab;

rotating the swab and at substantially the same time directing a sprayof charged droplets onto a surface of the swab in order to generate aplurality of analyte ions; and

analysing the analyte ions.

The step of rotating the swab whilst directing the spray of chargeddroplets onto the surface of the swab further may comprise substantiallycontinuously rotating the swab whilst directing the spray of chargeddroplets onto the surface of the swab.

The method may comprise translating and/or oscillating the swab whilstdirecting the spray of charged droplets onto the surface of the swab.

According to an aspect there is provided a method comprising:

providing a sample on a swab;

translating and/or oscillating the swab and at substantially the sametime directing a spray of charged droplets onto a surface of the swab inorder to generate a plurality of analyte ions; and

analysing the analyte ions.

The method may comprise substantially continuously translating and/oroscillating the swab whilst directing the spray of charged droplets ontothe surface of the swab.

The method may comprise translating and/or oscillating the swabsubstantially in the direction of the axial length of the swab.

According to an aspect there is provided apparatus comprising:

a first device arranged and adapted to direct a spray of chargeddroplets onto a surface of a swab in order to generate a plurality ofanalyte ions;

a second device arranged and adapted to rotate the swab at substantiallythe same time as the first device directs the spray of charged dropletsonto the surface of the swab; and

an analyser arranged and adapted to analyse the analyte ions.

The second device may be arranged and adapted to substantiallycontinuously rotate the swab whilst the first device directs the sprayof charged droplets onto the surface of the swab.

The apparatus may comprise a third device arranged and adapted totranslate and/or oscillate the swab whilst the first device directs thespray of charged droplets onto the surface of the swab.

According to an aspect there is provided apparatus comprising:

a first device arranged and adapted to direct a spray of chargeddroplets onto a surface of a swab in order to generate a plurality ofanalyte ions;

a third device arranged and adapted to translate and/or oscillate theswab whilst the first device directs the spray of charged droplets ontothe surface of the swab; and

an analyser arranged and adapted to analyse the analyte ions.

The third device may be arranged and adapted to substantiallycontinuously translate and/or oscillate the swab whilst the first devicedirects the spray of charged droplets onto the surface of the swab.

The third device may be arranged and adapted to translate and/oroscillate the swab substantially in the direction of the axial length ofthe swab.

According to an aspect there is provided a method comprising: providinga sample on a swab;

analysing the sample on the swab using a first analysis method; and

analysing the sample on the swab using one or more second differentanalysis methods;

wherein the first analysis method may comprise directing a spray ofcharged droplets onto a surface of the swab in order to generate aplurality of analyte ions and then analysing the analyte ions.

Directing the spray of charged droplets onto the swab may comprisedirecting a spray of charged solvent droplets onto the swab.

Directing the spray of charged droplets onto the swab may comprisedirecting the spray of charged droplets onto the swab at aboutatmospheric pressure.

Directing the spray of charged droplets onto the swab may compriseionising the sample using desorption electrospray ionisation (“DESI”) ordesorption electroflow focusing ionisation (“DEFFI”).

The one or more second analysis methods may comprise a culturinganalysis method.

The culturing analysis method may comprise contacting the swab with aculturing medium, incubating the culturing medium, and examining theculturing medium or a sample thereof under a microscope.

The one or more second analysis methods may comprise a gene sequencingmethod.

The sequencing method may comprise a 16S rRNA gene sequencing method.

The one or more second analysis methods may comprise a Matrix-AssistedLaser Desorption Ionisation (“MALDI”) method.

The one or more second analysis methods may comprise an ambientionisation mass spectrometry method.

The ambient ionisation mass spectrometry method may be selected from thegroup consisting of: (i) a rapid evaporative ionisation massspectrometry (“REIMS”) method; (ii) a laser desorption ionisation(“LDI”) method; (iii) a thermal desorption ionisation method; (iv) alaser diode thermal desorption (“LDTD”) ionisation method; (v) adesorption electro-flow focusing ionisation (“DEFFI”) method; (vi) adielectric barrier discharge (“DBD”) plasma ionisation method; (vii) anAtmospheric Solids Analysis Probe (“ASAP”) ionisation method; (viii) anultrasonic assisted spray ionisation method; (ix) an easy ambientsonic-spray ionisation (“EAST”) method; (x) a desorption atmosphericpressure photoionisation (“DAPPI”) method; (xi) a paperspray (“PS”)ionisation method; (xii) a jet desorption ionisation (“JeDI”) method;(xiii) a touch spray (“TS”) ionisation method; (xiv) a nano-DESIionisation method; (xv) a laser ablation electrospray (“LAESI”)ionisation method; (xvi) a direct analysis in real time (“DART”)ionisation method; (xvii) a probe electrospray ionisation (“PEST”)method; (xviii) a solid-probe assisted electrospray ionisation(“SPA-EST”) method; (xix) a cavitron ultrasonic surgical aspirator(“CUSA”) method; (xx) a focussed or unfocussed ultrasonic ablationmethod; (xxi) a microwave resonance method; and (xxii) a pulsed plasmaRF dissection method.

The method may comprise analysing the sample on the swab using a thirddifferent analysis method.

According to an aspect there is provided a method comprising:

providing a sample on a swab;

analysing the sample on the swab in a first mode of operation, whereinthe first mode of operation may comprise directing a spray of chargeddroplets onto a surface of the swab in order to generate a plurality ofanalyte ions; and

determining whether the analyte ions may comprise one or more ions ofinterest;

wherein if it is determined that the analyte ions comprise one or moreions of interest, then the method further may comprise:

analysing the sample on the swab in a second different mode ofoperation.

Directing the spray of charged droplets onto the swab may comprisedirecting a spray of charged solvent droplets onto the swab.

Directing the spray of charged droplets onto the swab may comprisedirecting the spray of charged droplets onto the swab at aboutatmospheric pressure.

Directing the spray of charged droplets onto the swab may compriseionising the sample using desorption electrospray ionisation (“DESI”) ordesorption electroflow focusing ionisation (“DEFFI”).

The second mode of operation may comprise directing a spray of chargeddroplets onto a surface of the swab in a second different mode ofoperation.

Either:

(i) the first mode of operation may comprise a positive ion mode ofoperation and the second mode of operation may comprise a negative ionmode of operation; or

(ii) the first mode of operation may comprise a negative ion mode ofoperation and the second mode of operation may comprise a positive ionmode of operation.

The first mode of operation may comprise directing a spray of chargeddroplets onto a surface of the swab, wherein the charged dropletscomprise a first solvent or solvent composition; and

the second mode of operation may comprise directing a spray of chargeddroplets onto a surface of the swab, wherein the charged dropletscomprise a second different solvent or solvent composition.

The second mode of operation may comprise an optimised version of thefirst mode of operation.

The second mode of operation may comprise generating a plurality ofanalyte ions from the sample using a second different ambient ionisationanalysis method.

The second different ambient ionisation analysis method may be selectedfrom the group consisting of: (i) a rapid evaporative ionisation massspectrometry (“REIMS”) method; (ii) a laser desorption ionisation(“LDI”) method; (iii) a thermal desorption ionisation method; (iv) alaser diode thermal desorption (“LDTD”) ionisation method; (v) adesorption electro-flow focusing ionisation (“DEFFI”) method; (vi) adielectric barrier discharge (“DBD”) plasma ionisation method; (vii) anAtmospheric Solids Analysis Probe (“ASAP”) ionisation method; (viii) anultrasonic assisted spray ionisation method; (ix) an easy ambientsonic-spray ionisation (“EAST”) method; (x) a desorption atmosphericpressure photoionisation (“DAPPI”) method; (xi) a paperspray (“PS”)ionisation method; (xii) a jet desorption ionisation (“JeDI”) method;(xiii) a touch spray (“TS”) ionisation method; (xiv) a nano-DESIionisation method; (xv) a laser ablation electrospray (“LAESI”)ionisation method; (xvi) a direct analysis in real time (“DART”)ionisation method; (xvii) a probe electrospray ionisation (“PEST”)method; (xviii) a solid-probe assisted electrospray ionisation(“SPA-EST”) method; (xix) a cavitron ultrasonic surgical aspirator(“CUSA”) method; (xx) a focussed or unfocussed ultrasonic ablationmethod; (xxi) a microwave resonance method; and (xxii) a pulsed plasmaRF dissection method.

The first mode of operation may comprise analysing the analyte ionsusing first operational parameters; and

the second mode of operation may comprise analysing analyte ions fromthe sample using second different operational parameters.

The first and/or second mode of operation may comprise (i) a mode ofoperation wherein analyte ions or ions derived from the analyte ions aremass analysed and/or ion mobility analysed; (ii) a mode of operationwherein the ion mobility, collision cross section or interaction crosssection of analyte ions or ions derived from the analyte ions may bedetermined; (iii) a mode of operation wherein analyte ions are furthersubjected to fragmentation; and/or (iv) a mode of operation whereinanalyte ions are reacted, excited, fragmented or fractionally separated.

The second different mode of operation may comprise: (i) a culturingmode of operation; (ii) a gene sequencing mode of operation; or (iii) aMatrix-Assisted Laser Desorption Ionisation (“MALDI”) mode of operation.

The method may comprise selecting and/or optimising the second mode ofoperation based on information acquired during the first mode ofoperation.

The method may comprise:

determining whether analyte ions analysed in the second mode ofoperation comprise one or more second ions of interest;

wherein if it is determined that the analyte ions comprise one or moresecond ions of interest, then the method further may comprise:

analysing the sample on the swab in a third different mode of operation.

According to an aspect there is provided apparatus comprising:

a first device arranged and adapted to analyse a sample on a swab in afirst mode of operation, wherein the first mode of operation maycomprise directing a spray of charged droplets onto a surface of theswab in order to generate a plurality of analyte ions; and

a second device arranged and adapted to determine whether the analyteions comprise one or more ions of interest, and if it is determined thatthe analyte ions comprise one or more ions of interest, to cause adevice to analyse the sample on the swab in a second different mode ofoperation.

The first device may be arranged and adapted to direct a spray ofcharged solvent droplets onto the swab.

The first device may be arranged and adapted to direct the spray ofcharged droplets onto the swab at about atmospheric pressure.

The first device may comprise a desorption electrospray ionisation(“DESI”) device or a desorption electroflow focusing ionisation(“DEFFI”) device.

The second device may be arranged and adapted to cause the first deviceto analyse the sample on the swab in a second different mode ofoperation.

Either:

(i) the first mode of operation may comprise a positive ion mode ofoperation and the second mode of operation may comprise a negative ionmode of operation; or

(ii) the first mode of operation may comprise a negative ion mode ofoperation and the second mode of operation may comprise a positive ionmode of operation.

In the first mode of operation the charged droplets may comprise a firstsolvent or solvent composition; and

in the second mode of operation the charged droplets may comprise asecond different solvent or solvent composition.

The second mode of operation may comprise an optimised version of thefirst mode of operation.

The second device may be arranged and adapted to cause a second deviceto analyse the sample on the swab using a second different ambientionisation analysis method.

The second device may be selected from the group consisting of: (i) arapid evaporative ionisation mass spectrometry (“REIMS”) ion source;(ii) a laser desorption ionisation (“LDI”) ion source; (iii) a thermaldesorption ionisation ion source; (iv) a laser diode thermal desorption(“LDTD”) ion source; (v) a desorption electro-flow focusing ionisation(“DEFFI”) ion source; (vi) a dielectric barrier discharge (“DBD”) plasmaion source; (vii) an Atmospheric Solids Analysis Probe (“ASAP”) ionsource; (viii) an ultrasonic assisted spray ion source; (ix) an easyambient sonic-spray ionisation (“EAST”) ion source; (x) a desorptionatmospheric pressure photoionisation (“DAPPI”) ion source; (xi) apaperspray (“PS”) ion source; (xii) a jet desorption ionisation (“JeDI”)ion source; (xiii) a touch spray (“TS”) ion source; (xiv) a nano-DESIion source; (xv) a laser ablation electrospray (“LAESI”) ion source;(xvi) a direct analysis in real time (“DART”) ion source; (xvii) a probeelectrospray ionisation (“PEST”) ion source; (xviii) a solid-probeassisted electrospray ionisation (“SPA-ESI”) ion source; (xix) acavitron ultrasonic surgical aspirator (“CUSA”) device; (xx) a focussedor unfocussed ultrasonic ablation device; (xxi) a microwave resonancedevice; and (xxii) a pulsed plasma RF dissection device.

The apparatus may be arranged and adapted in the first mode of operationto analyse the analyte ions using first operational parameters; and

the apparatus may be arranged and adapted in the second mode ofoperation to analyse analyte ions from the sample using second differentoperational parameters.

The apparatus may comprise: (i) a mass analyser or filter and/or ionmobility analyser for mass analysing and/or ion mobility analysinganalyte ions or ions derived from the analyte ions in the first and/orsecond mode of operation; (ii) an ion mobility device for determiningthe ion mobility, collision cross section or interaction cross sectionof analyte ions or ions derived from the analyte ions in the firstand/or second mode of operation; (iii) a fragmentation device forsubjecting analyte ions to fragmentation in the first and/or second modeof operation; and/or (iv) one or more devices for reacting, exciting,fragmenting and/or fractionally separating analyte ions in the firstand/or second mode of operation.

According to an aspect there is provided a method comprising:

automatically analysing a plurality of swabs by directing a spray ofcharged droplets onto a surface of each swab in order to generate aplurality of analyte ions; and

analysing the analyte ions from each swab.

A different sample may be provided on each of the plurality of swabs.

The step of automatically analysing the plurality of swabs may compriseautomatically analysing the plurality of swabs substantiallysequentially.

The step of automatically analysing the plurality of swabs may compriseautomatically analysing two or more of the plurality of swabssubstantially simultaneously or in parallel.

The method may comprise rotating, oscillating and/or translating eachswab and at substantially the same time directing the spray of chargeddroplets onto a surface of each swab in order to generate the pluralityof analyte ions.

According to an aspect there is provided apparatus comprising:

a first device arranged and adapted to automatically analyse a pluralityof swabs by directing a spray of charged droplets onto a surface of eachswab in order to generate a plurality of analyte ions; and

a second device arranged and adapted to analyse the analyte ions fromeach swab.

A different sample may be provided on each of the plurality of swabs.

The first device may be arranged and adapted to automatically analysethe plurality of swabs substantially sequentially.

The first device may be arranged and adapted to automatically analysetwo or more of the plurality of swabs substantially simultaneously or inparallel.

The apparatus may comprise a third device arranged and adapted torotate, oscillate and/or translate each swab whilst the first devicedirects the spray of charged droplets onto the surface of each swab.

According to an aspect there is provided a method comprising:

transferring a plurality of samples onto different positions on a roll,sheet, tape or substrate;

using an ambient ion source to analyse a first sample on the roll,sheet, tape or substrate;

advancing the roll, sheet, tape or substrate; and

using an ambient ion source to analyse a second sample on the roll,sheet, tape or substrate.

The method may comprise:

(i) advancing the roll, sheet, tape or substrate;

(ii) using an ambient ion source to analyse one or more further sampleson the roll, sheet, tape or substrate; and

(iii) optionally repeating steps (i) and (ii) one or more times.

The ambient ion source may comprise a desorption electrospray ionisation(“DESI”) ion source or a desorption electroflow focusing ionisation(“DEFFI”) ion source.

The method may comprise identifying the first sample and/or the secondsample and/or the one or more further samples.

According to an aspect there is provided a method comprising:

directing a spray of charged droplets onto a surface of a swab having asample of vaginal mucosa from a human or non-human animal providedthereon in order to generate a plurality of analyte ions;

analysing the analyte ions to obtain mass spectrometric data and/or ionmobility data; and

determining from the mass spectrometric data and/or ion mobility dataeither: (i) whether or not the human or non-human animal is pregnant;(ii) the stage or state of pregnancy of the human or non-human animal;(iii) whether or not the human or non-human animal is at an increasedrisk of an adverse pregnancy outcome; and/or (iv) whether or not thehuman or non-human animal is at an increased risk of having a pretermdelivery or a premature delivery.

The method may comprise identifying the presence of one or more microbesin the sample of vaginal mucosa, wherein the microbes are optionallybacteria.

The one or more microbes may be selected from the group consisting of:(i) Candida albicans; (ii) Pseudomonas montelli; (iii) Staphylococcusepidermis; (iv) Moraxella catarrhalis; (v) Klebsiella pneumonia; and(vi) Lactobacillus sp.

According to an aspect there is provided a method of pregnancy testingor pregnancy monitoring comprising a method as described above.

The method may comprise periodically repeating the method in order tomonitor the development of the pregnancy of the human or non-humananimal.

According to an aspect there is provided a method of diagnosis orprognosis comprising a method as described above.

According to an aspect there is provided a method comprising:

providing a faecal sample on an absorbent or other surface;

using an ambient ionisation source to generate a plurality of analyteions from the faecal sample; and

analysing the analyte ions.

The step of using an ambient ionisation source to generate the pluralityof analyte ions may comprise directing a spray of charged droplets ontothe absorbent or other surface.

The step of using an ambient ionisation source to generate the pluralityof analyte ions may comprise generating aerosol, smoke or vapour fromthe faecal sample.

The method may comprise ionising the aerosol, smoke or vapour in orderto generate the analyte ions.

The absorbent surface may comprise toilet tissue paper, tissue paper, anappy or diaper or an incontinence pad or pant.

The method may comprise determining based on the analysis whether or notthe faecal sample contains blood, human blood, non-human animal blood,haemoglobin, pathogens, undesired material, non-human material, parasitematerial or excreta or other waste products from parasites.

The method may comprise determining on the basis of whether or not thefaecal sample is determined to contain blood, human blood, non-humananimal blood or haemoglobin whether or not the human or non-human animalsuffers from or has an anal fissure, diverticular disease, aninflammatory disease, angiodysplasia and/or a polyp in their colon,bowel or other part of their body or suffers from or has another medicaldisease or condition.

The method may comprise analysing the level and/or composition of bilepresent in the faecal sample.

The method may comprise determining from the analysis of the leveland/or composition of bile present in the faecal sample whether or notthe human or non-human animal suffers from or has a liver disease,kidney disease or other medical disease or condition.

The method may comprise analysing the composition of thegastrointestinal microbiome of the human or non-human animal.

The method may comprise determining or assessing the impact ofantibiotics or probiotics upon the human or non-human animal based uponthe analysis of the gastrointestinal microbiome, in the faecal sample ofthe human or non-human animal.

The method may comprise determining, based on the analysis, whether thefaecal sample may comprise: (i) one or more particular microbes; (ii)one or more pathogens; (iii) one or more parasites; and/or (iv) one ormore metabolites.

According to an aspect there is provided the use of a swab as describedabove in a method according as described above.

According to an aspect there is provided a method of desorptionelectrospray ionisation (“DESI”) comprising a method as described above.

According to an aspect there is provided a method of mass spectrometrycomprising a method as described above.

According to an aspect there is provided a desorption electrosprayionisation (“DESI”) ion source comprising apparatus as described above.

According to an aspect there is provided a mass and/or ion mobilityspectrometer comprising apparatus as described above.

According to an aspect there is provided a mass and/or ion mobilityspectrometer comprising:

a first device arranged and adapted to accommodate a biopsy sample;

a second device arranged and adapted to generate analyte ions from abiopsy sample within the first device, wherein the second device may bearranged and adapted to generate first analyte ions from a firstposition on the biopsy sample at a first time, and to generate secondanalyte ions from a second different position on the biopsy sample at asecond different time; and

an analyser arranged and adapted to analyse the analyte ions.

The biopsy sample may comprise a sample of tissue having a longitudinallength.

The composition of the sample of tissue may vary or change along thelongitudinal length.

The longitudinal length may correspond to the depth within a tissue.

The biopsy sample may comprise a biopsy core or cylinder.

The first device may comprise a channel arranged and adapted toaccommodate a biopsy core.

The second device may be arranged and adapted to generate first analyteions from a first position along the longitudinal length of the biopsysample at the first time, and to generate second analyte ions from asecond different position along the longitudinal length of the biopsysample at the second different time.

The second device may be arranged and adapted to scan at least a portionof the longitudinal length of the biopsy sample so as to generateanalyte ions from multiple positions along the longitudinal length ofthe biopsy sample.

The second device may comprise an ambient ionisation ion source.

The ambient ionisation ion source may comprise an ion source selectedfrom the group consisting of: (i) a rapid evaporative ionisation massspectrometry (“REIMS”) ion source; (ii) a desorption electrosprayionisation (“DESI”) ion source; (iii) a laser desorption ionisation(“LDI”) ion source; (iv) a thermal desorption ion source; (v) a laserdiode thermal desorption (“LDTD”) ion source; (vi) a desorptionelectro-flow focusing (“DEFFI”) ion source; (vii) a dielectric barrierdischarge (“DBD”) plasma ion source; (viii) an Atmospheric SolidsAnalysis Probe (“ASAP”) ion source; (ix) an ultrasonic assisted sprayionisation ion source; (x) an easy ambient sonic-spray ionisation(“EAST”) ion source; (xi) a desorption atmospheric pressurephotoionisation (“DAPPI”) ion source; (xii) a paperspray (“PS”) ionsource; (xiii) a jet desorption ionisation (“JeDI”) ion source; (xiv) atouch spray (“TS”) ion source; (xv) a nano-DESI ion source; (xvi) alaser ablation electrospray (“LAESI”) ion source; (xvii) a directanalysis in real time (“DART”) ion source; (xviii) a probe electrosprayionisation (“PEST”) ion source; (xix) a solid-probe assistedelectrospray ionisation (“SPA-EST”) ion source; (xx) a cavitronultrasonic surgical aspirator (“CUSA”) device; (xxi) a focussed orunfocussed ultrasonic ablation device; (xxii) a microwave resonancedevice; and (xxiii) a pulsed plasma RF dissection device.

The second device may be arranged and adapted to generate aerosol, smokeor vapour from the biopsy sample, and to ionise the aerosol, smoke orvapour in order to generate the analyte ions.

The second device may comprise one or more electrodes arranged andadapted to contact the biopsy sample to generate the aerosol, smoke orvapour.

The one or more electrodes may comprise a bipolar device or a monopolardevice.

The one or more electrodes may comprise either: (i) a monopolar device,wherein the apparatus optionally further comprises a separate returnelectrode; (ii) a bipolar device; or (iii) a multi-phase RF device,wherein the apparatus optionally further comprises a separate returnelectrode or electrodes.

The one or more electrodes may comprise a rapid evaporation ionisationmass spectrometry (“REIMS”) device.

The mass and/or ion mobility spectrometer may comprise a device arrangedand adapted to apply an AC or RF voltage to the one or more electrodesin order to generate the aerosol, smoke or vapour.

The device for applying the AC or RF voltage to the one or moreelectrodes may be arranged and adapted to apply one or more pulses ofthe AC or RF voltage to the one or more electrodes.

Application of the AC or RF voltage to the one or more electrodes maycause heat to be dissipated into the sample.

The second device may comprise a laser for irradiating the sample.

The second device may be arranged and adapted to generate aerosol, smokeor vapour from the sample by direct evaporation or vaporisation ofsample material from the sample by Joule heating or diathermy.

The second device may be arranged and adapted to direct ultrasonicenergy into the sample.

The second device may be arranged and adapted to direct a spray ofcharged droplets onto the biopsy sample so as to generate the analyteions.

The ambient ionisation ion source may comprise a desorption electrosprayionisation (“DESI”) ion source or a desorption electroflow focusingionisation (“DEFFI”) ion source.

The desorption electrospray ionisation (“DESI”) ion source or desorptionelectroflow focusing ionisation (“DEFFI”) ion source may comprise agradient desorption electrospray ionisation (“DESI”) ion source or agradient desorption electroflow focusing ionisation (“DEFFI”) ion sourcewherein the composition of a solvent supplied to and/or emitted from thedesorption electrospray ionisation (“DESI”) ion source or desorptionelectroflow focusing ionisation (“DEFFI”) ion source is varied as afunction of time.

The desorption electrospray ionisation (“DESI”) ion source or desorptionelectroflow focusing ionisation (“DEFFI”) ion source may be arranged andadapted to perform a gradient desorption electrospray ionisationanalysis of the biopsy sample.

The desorption electrospray ionisation (“DESI”) ion source or desorptionelectroflow focusing ionisation (“DEFFI”) ion source may be arranged andadapted to perform a gradient desorption electrospray ionisationanalysis along the length of the biopsy sample, wherein the compositionof a solvent supplied to and/or emitted from the desorption electrosprayionisation (“DESI”) ion source or desorption electroflow focusingionisation (“DEFFI”) ion source is varied as a function of positionalong the length of the biopsy sample.

The analyser may comprise: (i) a mass analyser or filter and/or ionmobility analyser for mass analysing and/or ion mobility analysing theanalyte ions and/or ions derived from the analyte ions; (ii) an ionmobility device for determining the ion mobility, collision crosssection or interaction cross section of the analyte ions and/or ionsderived from the analyte ions; and/or (iii) one or more fragmentation,collision or reaction devices for fragmenting or reacting the analyteions.

According to an aspect there is provided a method of mass spectrometrycomprising: providing a biopsy sample;

generating first analyte ions from a first position on the biopsy sampleat a first time, and generating second analyte ions from a seconddifferent position on the biopsy sample at a second different time; and

analysing the analyte ions.

The biopsy sample may comprise a sample of tissue having a longitudinallength.

The composition of the sample of tissue may vary or change along thelongitudinal length.

The longitudinal length may correspond to the depth within a tissue.

The biopsy sample may comprise a biopsy core or cylinder.

The method may comprise accommodating the biopsy sample in a channel.

The method may comprise generating first analyte ions from a firstposition along the longitudinal length of the biopsy sample at the firsttime, and generating second analyte ions from a second differentposition along the longitudinal length of the biopsy sample at thesecond different time.

The method may comprise scanning at least a portion of the longitudinallength of the biopsy sample so as to generate analyte ions from multiplepositions along the longitudinal length of the biopsy sample.

The method may comprise generating the analyte ions using an ambientionisation ion source.

The ambient ionisation ion source may comprise an ion source selectedfrom the group consisting of: (i) a rapid evaporative ionisation massspectrometry (“REIMS”) ion source; (ii) a desorption electrosprayionisation (“DESI”) ion source; (iii) a laser desorption ionisation(“LDI”) ion source; (iv) a thermal desorption ion source; (v) a laserdiode thermal desorption (“LDTD”) ion source; (vi) a desorptionelectro-flow focusing (“DEFFI”) ion source; (vii) a dielectric barrierdischarge (“DBD”) plasma ion source; (viii) an Atmospheric SolidsAnalysis Probe (“ASAP”) ion source; (ix) an ultrasonic assisted sprayionisation ion source; (x) an easy ambient sonic-spray ionisation(“EAST”) ion source; (xi) a desorption atmospheric pressurephotoionisation (“DAPPI”) ion source; (xii) a paperspray (“PS”) ionsource; (xiii) a jet desorption ionisation (“JeDI”) ion source; (xiv) atouch spray (“TS”) ion source; (xv) a nano-DESI ion source; (xvi) alaser ablation electrospray (“LAESI”) ion source; (xvii) a directanalysis in real time (“DART”) ion source; (xviii) a probe electrosprayionisation (“PEST”) ion source; (xix) a solid-probe assistedelectrospray ionisation (“SPA-EST”) ion source; (xx) a cavitronultrasonic surgical aspirator (“CUSA”) device; (xxi) a focussed orunfocussed ultrasonic ablation device; (xxii) a microwave resonancedevice; and (xxiii) a pulsed plasma RF dissection device.

The method may comprise generating aerosol, smoke or vapour from thebiopsy sample, and ionising the aerosol, smoke or vapour in order togenerate the analyte ions.

The method may comprise contacting the biopsy sample with one or moreelectrodes to generate the aerosol, smoke or vapour.

The one or more electrodes may comprise a bipolar device or a monopolardevice.

The one or more electrodes may comprise either: (i) a monopolar device,wherein the apparatus optionally further comprises a separate returnelectrode; (ii) a bipolar device; or (iii) a multi-phase RF device,wherein the apparatus optionally further comprises a separate returnelectrode or electrodes.

The one or more electrodes may comprise a rapid evaporation ionisationmass spectrometry (“REIMS”) device.

The method may comprise applying an AC or RF voltage to the one or moreelectrodes in order to generate the aerosol, smoke or vapour.

The step of applying the AC or RF voltage to the one or more electrodesmay comprise applying one or more pulses of the AC or RF voltage to theone or more electrodes.

Applying the AC or RF voltage to the one or more electrodes may causeheat to be dissipated into the sample.

The method may comprise irradiating the sample with a laser in order togenerate the analyte ions.

The method may comprise directing ultrasonic energy into the sample.

The method may comprise directing a spray of charged droplets onto thebiopsy sample so as to generate the analyte ions.

The step of directing the spray of charged droplets onto the biopsysample may comprise using a desorption electrospray ionisation (“DESI”)ion source or a desorption electroflow focusing ionisation (“DEFFI”) ionsource to generate the analyte ions.

The method may comprise varying the composition of a solvent supplied toand/or emitted from the desorption electrospray ionisation (“DESI”) ionsource or desorption electroflow focusing ionisation (“DEFFI”) ionsource as a function of time.

The method may comprise performing a gradient desorption electrosprayionisation analysis of the biopsy sample.

The method may comprise varying the composition of a solvent supplied toand/or emitted from the desorption electrospray ionisation (“DESI”) ionsource or desorption electroflow focusing ionisation (“DEFFI”) ionsource as a function of position along the length of the biopsy sample.

The method may comprise: (i) mass analysing and/or ion mobilityanalysing the analyte ions and/or ions derived from the analyte ions;(ii) determining the ion mobility, collision cross section orinteraction cross section of the analyte ions and/or ions derived fromthe analyte ions; and/or (iii) fragmenting or reacting the analyte ions.

The method may comprise analysing a disease.

The method may comprise determining the presence, location, marginand/or size of a tumour.

The method may comprise characterising a tumour based on: (i) theaggressive of the tumour; (ii) the susceptibility of the tumour totreatment; (iii) whether and/or how much of the tumour can be surgicallyremoved; and/or (iv) whether and/or how much of the tumour can beremoved based on the location of the tumour.

According to an aspect there is provided a method comprising:

sampling tissue using a biopsy needle so as to produce a first biopsysample and a second biopsy sample;

analysing the first biopsy sample in a first mode of operation, whereinthe first mode of operation may comprise generating analyte ions fromthe first biopsy sample and analysing the analyte ions; and

analysing the second biopsy sample in a second different mode ofoperation.

The first mode of operation may comprise generating analyte ions fromthe first biopsy sample using a first ambient ionisation analysismethod.

The first ambient ionisation analysis method may be selected from thegroup consisting of: (i) a rapid evaporative ionisation massspectrometry (“REIMS”) method; (ii) a desorption electrospray ionisation(“DESI”) ionisation method; (iii) a laser desorption ionisation (“LDI”)method; (iv) a thermal desorption ionisation method; (v) a laser diodethermal desorption (“LDTD”) ionisation method; (vi) a desorptionelectro-flow focusing ionisation (“DEFFI”) method; (vii) a dielectricbarrier discharge (“DBD”) plasma ionisation method; (viii) anAtmospheric Solids Analysis Probe (“ASAP”) ionisation method; (ix) anultrasonic assisted spray ionisation method; (x) an easy ambientsonic-spray ionisation (“EAST”) method; (xi) a desorption atmosphericpressure photoionisation (“DAPPI”) method; (xii) a paperspray (“PS”)ionisation method; (xiii) a jet desorption ionisation (“JeDI”) method;(xiv) a touch spray (“TS”) ionisation method; (xv) a nano-DESIionisation method; (xvi) a laser ablation electrospray (“LAESI”)ionisation method; (xvii) a direct analysis in real time (“DART”)ionisation method; (xviii) a probe electrospray ionisation (“PEST”)method; (xix) a solid-probe assisted electrospray ionisation (“SPA-EST”)method; (xx) a cavitron ultrasonic surgical aspirator (“CUSA”) method;(xxi) a focussed or unfocussed ultrasonic ablation method; (xxii) amicrowave resonance method; and (xxiii) a pulsed plasma RF dissectionmethod.

The step of generating analyte ions from the first biopsy sample maycomprise directing a spray of charged droplets onto the first biopsysample in order to generate the analyte ions.

Directing the spray of charged droplets onto the first biopsy sample maycomprise directing a spray of charged solvent droplets onto the firstbiopsy sample.

Directing the spray of charged droplets onto the first biopsy sample maycomprise directing the spray of charged droplets onto the first biopsysample at about atmospheric pressure.

Directing the spray of charged droplets onto the first biopsy sample maycomprise ionising the sample using desorption electrospray ionisation(“DESI”) or desorption electroflow focusing ionisation (“DEFFI”).

The step of generating analyte ions from the first biopsy sample maycomprise generating aerosol, smoke or vapour from the first biopsysample, and ionising the aerosol, smoke or vapour to generate theanalyte ions.

The step of generating analyte ions from the first biopsy sample maycomprise contacting one or more electrodes to the biopsy sample togenerate the aerosol, smoke or vapour.

The one or more electrodes may comprise a bipolar device or a monopolardevice.

The one or more electrodes may comprise either: (i) a monopolar device,wherein the apparatus optionally further comprises a separate returnelectrode; (ii) a bipolar device; or (iii) a multi-phase RF device,wherein the apparatus optionally further comprises a separate returnelectrode or electrodes.

The one or more electrodes may comprise a rapid evaporation ionisationmass spectrometry (“REIMS”) device.

The method may comprise applying an AC or RF voltage to the one or moreelectrodes in order to generate the aerosol, smoke or vapour.

The step of applying the AC or RF voltage to the one or more electrodesmay comprise applying one or more pulses of the AC or RF voltage to theone or more electrodes.

Applying the AC or RF voltage to the one or more electrodes may causeheat to be dissipated into the sample.

The step of generating analyte ions from the first biopsy sample maycomprise directing a laser beam onto the first biopsy sample to generatethe analyte ions.

The second mode of operation may comprise generating analyte ions fromthe second biopsy sample and analysing the analyte ions in a seconddifferent mode of operation.

The second mode of operation may comprise generating analyte ions fromthe second biopsy sample using the first ambient ionisation analysismethod in a second different mode of operation.

Either:

(i) the first mode of operation may comprise a positive ion mode ofoperation and the second mode of operation may comprise a negative ionmode of operation; or

(ii) the first mode of operation may comprise a negative ion mode ofoperation and the second mode of operation may comprise a positive ionmode of operation.

The first mode of operation may comprise generating the analyte ionsfrom the first biopsy sample using a first solvent or solventcomposition; and

the second mode of operation may comprise generating the analyte ionsfrom the second biopsy sample using a second different solvent orsolvent composition.

The second mode of operation may comprise an optimised version of thefirst mode of operation.

The second mode of operation may comprise generating a plurality ofanalyte ions from the second biopsy sample using a second differentambient ionisation analysis method.

The second different ambient ionisation analysis method may be selectedfrom the group consisting of: (i) a rapid evaporative ionisation massspectrometry (“REIMS”) method; (ii) a desorption electrospray ionisation(“DESI”) ionisation method; (iii) a laser desorption ionisation (“LDI”)method; (iv) a thermal desorption ionisation method; (v) a laser diodethermal desorption (“LDTD”) ionisation method; (vi) a desorptionelectro-flow focusing ionisation (“DEFFI”) method; (vii) a dielectricbarrier discharge (“DBD”) plasma ionisation method; (viii) anAtmospheric Solids Analysis Probe (“ASAP”) ionisation method; (ix) anultrasonic assisted spray ionisation method; (x) an easy ambientsonic-spray ionisation (“EAST”) method; (xi) a desorption atmosphericpressure photoionisation (“DAPPI”) method; (xii) a paperspray (“PS”)ionisation method; (xiii) a jet desorption ionisation (“JeDI”) method;(xiv) a touch spray (“TS”) ionisation method; (xv) a nano-DESIionisation method; (xvi) a laser ablation electrospray (“LAESI”)ionisation method; (xvii) a direct analysis in real time (“DART”)ionisation method; (xviii) a probe electrospray ionisation (“PEST”)method; (xix) a solid-probe assisted electrospray ionisation (“SPA-EST”)method; (xx) a cavitron ultrasonic surgical aspirator (“CUSA”) method;(xxi) a focussed or unfocussed ultrasonic ablation method; (xxii) amicrowave resonance method; and (xxiii) a pulsed plasma RF dissectionmethod.

The first mode of operation may comprise analysing the analyte ions fromthe first biopsy sample using first operational parameters; and

the second mode of operation may comprise analysing the analyte ionsfrom the second biopsy sample using second different operationalparameters.

The first and/or second mode of operation may comprise: (i) a mode ofoperation wherein analyte ions or ions derived from the analyte ions aremass analysed and/or ion mobility analysed; (ii) a mode of operationwherein the ion mobility, collision cross section or interaction crosssection of analyte ions or ions derived from the analyte ions isdetermined; (iii) a mode of operation wherein analyte ions are subjectedto fragmentation; and/or (iv) a mode of operation wherein analyte ionsare reacted, excited, fragmented or fractionally separated.

The second different mode of operation may comprise: (i) a genesequencing mode of operation; (ii) a Matrix-Assisted Laser DesorptionIonisation (“MALDI”) mode of operation; and/or (iii) a histopathologicalmode of operation.

The method may comprise selecting and/or optimising the second mode ofoperation based on information acquired during the first mode ofoperation.

The first biopsy sample may comprise a first portion of the tissuehaving a first longitudinal length and/or the second biopsy sample maycomprise a second portion of the tissue having a second longitudinallength.

The composition of the first biopsy sample may vary or change along thefirst longitudinal length and/or the composition of the second biopsysample may vary or change along the second longitudinal length.

The first longitudinal length may correspond to the depth within thetissue and/or the second longitudinal length may correspond to the depthwithin the tissue.

The first biopsy sample may comprise a biopsy core or cylinder and/orthe second biopsy sample may comprise a biopsy core or cylinder.

The first biopsy sample may comprise a first portion of the tissue andthe second biopsy sample may comprise a second portion of the tissue;and

the first portion of the tissue may have been adjacent and/or connectedto the second portion of the tissue.

The first portion of the tissue may have been adjacent and/or connectedto the second portion of the tissue along some, most or all the axiallength of the first and/or second portion of the tissue.

Sampling tissue using the biopsy needle may comprise producing the firstbiopsy sample and the second biopsy sample substantially at the sametime.

Sampling tissue using the biopsy needle may comprise inserting thebiopsy needle into the tissue a single time so as to produce the firstand second samples.

The biopsy needle may comprise a needle comprising a first hollow tubeor cylinder and a second hollow tube or cylinder.

The first hollow tube or cylinder and second hollow tube or cylinder maybe conjoined.

The first hollow tube or cylinder and second hollow tube or cylinder maybe conjoined along some, most or all of an axial length of the firstand/or an axial length of the second hollow tube or cylinder.

According to an aspect there is provided a biopsy needle arranged andadapted to produce a first biopsy sample and a second biopsy sample whensampling tissue.

The first biopsy sample may comprise a first portion of the tissuehaving a first longitudinal length and/or the second biopsy sample maycomprise a second portion of the tissue having a second longitudinallength.

The composition of the first biopsy sample may vary or change along thefirst longitudinal length and/or the composition of the second biopsysample may vary or change along the second longitudinal length.

The first longitudinal length may correspond to the depth within thetissue and/or the second longitudinal length may correspond to the depthwithin the tissue.

The first biopsy sample may comprise a biopsy core or cylinder and/orthe second biopsy sample may comprise a biopsy core or cylinder.

The first biopsy sample may comprise a first portion of the tissue andthe second biopsy sample may comprise a second portion of the tissue;

wherein the first portion of the tissue may have been adjacent and/orconnected to the second portion of the tissue.

The first portion of the tissue may have been adjacent and/or connectedto the second portion of the tissue along some, most or all the axiallength of the first and/or second portion of the tissue.

The biopsy needle may be arranged and adapted to produce the first andsecond samples substantially at the same time.

The biopsy needle may be arranged and adapted to produce the first andsecond samples when inserted into the tissue a single time.

The biopsy needle may comprise a needle comprising a first hollow tubeor cylinder and a second hollow tube or cylinder.

The first hollow tube or cylinder and second hollow tube or cylinder maybe conjoined.

The first hollow tube or cylinder and second hollow tube or cylinder maybe conjoined along some, most or all of an axial length of the firstand/or an axial length of the second hollow tube or cylinder.

According to an aspect there is provided apparatus comprising:

a biopsy needle comprising one or more ambient ionisation devices;

a control system arranged and adapted to energise the one or moreambient ionisation devices in order to generate aerosol, smoke or vapourfrom a biopsy sample within the biopsy needle; and

an analyser for analysing the aerosol, smoke or vapour.

The one or more ambient ionisation devices may comprise one or moreelectrodes arranged and adapted to contact the biopsy sample to generatethe aerosol, smoke or vapour.

The one or more electrodes may comprise a bipolar device or a monopolardevice.

The one or more electrodes may comprise either: (i) a monopolar device,wherein the apparatus optionally further comprises a separate returnelectrode; (ii) a bipolar device; or (iii) a multi-phase RF device,wherein the apparatus optionally further comprises a separate returnelectrode or electrodes.

The one or more electrodes may comprise a rapid evaporation ionisationmass spectrometry (“REIMS”) device.

The control system may be arranged and adapted to apply an AC or RFvoltage to the one or more electrodes in order to generate the aerosol,smoke or vapour.

The control system may be arranged and adapted to apply one or morepulses of the AC or RF voltage to the one or more electrodes.

Application of the AC or RF voltage to the one or more electrodes maycause heat to be dissipated into the sample.

The one or more ambient ionisation devices may comprise a laser forirradiating the sample.

The one or more ambient ionisation devices may be arranged and adaptedto generate aerosol, smoke or vapour from the sample by directevaporation or vaporisation of sample material from the sample by Jouleheating or diathermy.

The analyser may comprise a collision surface, and wherein the apparatusmay be arranged and adapted to cause at least some of the aerosol, smokeand/or vapour to impact upon the collision surface in order to formanalyte ions.

The analyser may comprise: (i) a mass analyser or filter and/or ionmobility analyser for mass analysing and/or ion mobility analysing theaerosol, smoke, vapour, or the analyte ions and/or ions derived from theaerosol, smoke, vapour, the analyte ions; (ii) an ion mobility devicefor determining the ion mobility, collision cross section or interactioncross section of the aerosol, smoke, vapour, or the analyte ions and/orions derived from the aerosol, smoke, vapour, the analyte ions; and/or(iii) one or more fragmentation, collision or reaction devices forfragmenting or reacting the aerosol, smoke, vapour, or the analyte ions.

According to an aspect there is provided a biopsy needle comprising oneor more ambient ionisation devices.

The one or more ambient ionisation devices may comprise one or moreelectrodes arranged and adapted to contact a biopsy sample within thebiopsy needle to generate aerosol, smoke or vapour.

The one or more electrodes may comprise a bipolar device or a monopolardevice.

The one or more electrodes may comprise either: (i) a monopolar device,wherein the apparatus optionally further comprises a separate returnelectrode; (ii) a bipolar device; or (iii) a multi-phase RF device,wherein the apparatus optionally further comprises a separate returnelectrode or electrodes.

The one or more electrodes may comprise a rapid evaporation ionisationmass spectrometry (“REIMS”) device.

The one or more ambient ionisation devices may comprise a laser forirradiating a biopsy sample within the biopsy needle.

According to an aspect there is provided a method comprising:

providing a biopsy needle comprising one or more ambient ionisationdevices;

energising the one or more ambient ionisation devices in order togenerate aerosol, smoke or vapour from a biopsy sample within the biopsyneedle; and

analysing the aerosol, smoke or vapour.

The one or more ambient ionisation devices may comprise one or moreelectrodes arranged and adapted to contact the biopsy sample to generatethe aerosol, smoke or vapour.

The one or more electrodes may comprise a bipolar device or a monopolardevice.

The one or more electrodes may comprise either: (i) a monopolar device,wherein the apparatus optionally further comprises a separate returnelectrode; (ii) a bipolar device; or (iii) a multi-phase RF device,wherein the apparatus optionally further comprises a separate returnelectrode or electrodes.

The one or more electrodes may comprise a rapid evaporation ionisationmass spectrometry (“REIMS”) device.

Energising the one or more ambient ionisation devices may compriseapplying an AC or RF voltage to the one or more electrodes in order togenerate the aerosol, smoke or vapour.

Applying the AC or RF voltage to the one or more electrodes may compriseapplying one or more pulses of the AC or RF voltage to the one or moreelectrodes.

Applying the AC or RF voltage to the one or more electrodes may causeheat to be dissipated into the sample.

The one or more ambient ionisation devices may comprise a laser forirradiating the sample.

The method may comprise generating the aerosol, smoke or vapour from thesample by direct evaporation or vaporisation of sample material from thesample by Joule heating or diathermy.

The method may comprise causing at least some of the aerosol, smokeand/or vapour to impact upon a collision surface in order to formanalyte ions.

Analysing the aerosol, smoke or vapour may comprise: (i) mass analysingand/or ion mobility analysing the aerosol, smoke, vapour, or the analyteions and/or ions derived from the aerosol, smoke, vapour, the analyteions; (ii) determining the ion mobility, collision cross section orinteraction cross section of the aerosol, smoke, vapour, or the analyteions and/or ions derived from the aerosol, smoke, vapour, the analyteions; and/or (iii) fragmenting or reacting the aerosol, smoke, vapour,or the analyte ions.

The method may comprise inserting the biopsy needle into a tissue so asto provide the biopsy sample within the biopsy needle.

The method may comprise energising the one or more ambient ionisationdevices when the biopsy sample is inserted into the tissue.

According to an aspect there is provided a method comprising:

sampling tissue to produce one or more biopsy samples;

analysing the one or more biopsy samples; and

performing a diagnostic or surgical procedure using a first device thatmay comprise generating analyte ions from tissue and analysing theanalyte ions, wherein one or more operational parameters of the firstdevice are calibrated, optimised or varied on the basis of the analysisof the one or more biopsy samples.

One or more of the one or more biopsy samples may comprise a sample oftissue having a longitudinal length.

The composition of the sample of tissue may vary or change along thelongitudinal length.

The longitudinal length may correspond to the depth within a tissue.

One or more of the one or more biopsy samples may comprise a biopsy coreor cylinder.

Analysing the one or more biopsy samples may comprise generating analyteions from the one or more biopsy samples and analysing the analyte ions.

Generating analyte ions from the one or more biopsy samples may comprisegenerating analyte ions from the one or more biopsy samples using anambient ionisation ion source.

The ambient ionisation ion source may comprise an ion source selectedfrom the group consisting of: (i) a rapid evaporative ionisation massspectrometry (“REIMS”) ion source; (ii) a desorption electrosprayionisation (“DESI”) ion source; (iii) a laser desorption ionisation(“LDI”) ion source; (iv) a thermal desorption ion source; (v) a laserdiode thermal desorption (“LDTD”) ion source; (vi) a desorptionelectro-flow focusing (“DEFFI”) ion source; (vii) a dielectric barrierdischarge (“DBD”) plasma ion source; (viii) an Atmospheric SolidsAnalysis Probe (“ASAP”) ion source; (ix) an ultrasonic assisted sprayionisation ion source; (x) an easy ambient sonic-spray ionisation(“EASI”) ion source; (xi) a desorption atmospheric pressurephotoionisation (“DAPPI”) ion source; (xii) a paperspray (“PS”) ionsource; (xiii) a jet desorption ionisation (“JeDI”) ion source; (xiv) atouch spray (“TS”) ion source; (xv) a nano-DESI ion source; (xvi) alaser ablation electrospray (“LAESI”) ion source; (xvii) a directanalysis in real time (“DART”) ion source; (xviii) a probe electrosprayionisation (“PESI”) ion source; (xix) a solid-probe assistedelectrospray ionisation (“SPA-EST”) ion source; (xx) a cavitronultrasonic surgical aspirator (“CUSA”) device; (xxi) a focussed orunfocussed ultrasonic ablation device; (xxii) a microwave resonancedevice; and (xxiii) a pulsed plasma RF dissection device.

The step of generating analyte ions from the one or more biopsy samplesmay comprise generating aerosol, smoke or vapour from the one or morebiopsy samples, and ionising the aerosol, smoke or vapour to generatethe analyte ions.

The method may comprise causing at least some of the aerosol, smokeand/or vapour to impact upon a collision surface in order to generatethe analyte ions.

The step of generating analyte ions from the one or more biopsy samplesmay comprise contacting one or more electrodes to the one or more biopsysamples to generate the aerosol, smoke or vapour.

The one or more electrodes may comprise a bipolar device or a monopolardevice.

The one or more electrodes may comprise either: (i) a monopolar device,wherein the apparatus optionally further comprises a separate returnelectrode; (ii) a bipolar device; or (iii) a multi-phase RF device,wherein the apparatus optionally further comprises a separate returnelectrode or electrodes.

The one or more electrodes may comprise a rapid evaporation ionisationmass spectrometry (“REIMS”) device.

The method may comprise applying an AC or RF voltage to the one or moreelectrodes in order to generate the aerosol, smoke or vapour.

The step of applying the AC or RF voltage to the one or more electrodesmay comprise applying one or more pulses of the AC or RF voltage to theone or more electrodes.

Applying the AC or RF voltage to the one or more electrodes may causeheat to be dissipated into the sample.

The step of generating analyte ions from the one or more biopsy samplesmay comprise directing a laser beam onto the one or more biopsy samplesto generate the analyte ions.

Analysing analyte ions may comprise: (i) mass analysing and/or ionmobility analysing the analyte ions and/or ions derived from the analyteions; (ii) determining the ion mobility, collision cross section orinteraction cross section of the analyte ions and/or ions derived fromthe analyte ions; and/or (iii) fragmenting or reacting the analyte ions.

Analysing the one or more biopsy samples may comprise analysing the oneor more biopsy samples using the first device.

The first device may comprise an ambient ionisation ion source.

The ambient ionisation ion source may comprise an ion source selectedfrom the group consisting of: (i) a rapid evaporative ionisation massspectrometry (“REIMS”) ion source; (ii) a desorption electrosprayionisation (“DESI”) ion source; (iii) a laser desorption ionisation(“LDI”) ion source; (iv) a thermal desorption ion source; (v) a laserdiode thermal desorption (“LDTD”) ion source; (vi) a desorptionelectro-flow focusing (“DEFFI”) ion source; (vii) a dielectric barrierdischarge (“DBD”) plasma ion source; (viii) an Atmospheric SolidsAnalysis Probe (“ASAP”) ion source; (ix) an ultrasonic assisted sprayionisation ion source; (x) an easy ambient sonic-spray ionisation(“EAST”) ion source; (xi) a desorption atmospheric pressurephotoionisation (“DAPPI”) ion source; (xii) a paperspray (“PS”) ionsource; (xiii) a jet desorption ionisation (“JeDI”) ion source; (xiv) atouch spray (“TS”) ion source; (xv) a nano-DESI ion source; (xvi) alaser ablation electrospray (“LAESI”) ion source; (xvii) a directanalysis in real time (“DART”) ion source; (xviii) a probe electrosprayionisation (“PESI”) ion source; (xix) a solid-probe assistedelectrospray ionisation (“SPA-EST”) ion source; (xx) a cavitronultrasonic surgical aspirator (“CUSA”) device; (xxi) a focussed orunfocussed ultrasonic ablation device; (xxii) a microwave resonancedevice; and (xxiii) a pulsed plasma RF dissection device.

The step of performing a diagnostic or surgical procedure may compriseusing the first device to generate aerosol, smoke or vapour from thetissue, and ionising the aerosol, smoke or vapour in order to generatethe analyte ions.

The method may comprise causing at least some of the aerosol, smoke orvapour to impact upon a collision surface in order to generate theanalyte ions.

Using the first device to generate aerosol, smoke or vapour from thetissue may comprise contacting the tissue with one or more electrodes togenerate the aerosol, smoke or vapour.

The one or more electrodes may comprise a bipolar device or a monopolardevice.

The one or more electrodes may comprise either: (i) a monopolar device,wherein the apparatus optionally further comprises a separate returnelectrode; (ii) a bipolar device; or (iii) a multi-phase RF device,wherein the apparatus optionally further comprises a separate returnelectrode or electrodes.

The method may comprise applying an AC or RF voltage to the one or moreelectrodes in order to generate the aerosol, smoke or vapour.

Applying the AC or RF voltage to the one or more electrodes may compriseapplying one or more pulses of the AC or RF voltage to the one or moreelectrodes.

Applying the AC or RF voltage to the one or more electrodes may causeheat to be dissipated into the sample.

The first device and/or the one or more electrodes may comprise a rapidevaporation ionisation mass spectrometry (“REIMS”) device.

The first device may comprise a laser for irradiating the sample.

Analysing the analyte ions may comprise: (i) mass analysing and/or ionmobility analysing the analyte ions and/or ions derived from the analyteions; (ii) determining the ion mobility, collision cross section orinteraction cross section of the analyte ions and/or ions derived fromthe analyte ions; and/or (iii) fragmenting or reacting the analyte ions.

The first device may comprise an electrosurgical tool.

The first device may comprise an electrosurgical device, a diathermydevice, an ultrasonic device, hybrid ultrasonic electrosurgical device,surgical water jet device, hybrid electrosurgery, argon plasmacoagulation device, hybrid argon plasma coagulation device and water jetdevice and/or a laser device.

Analysing the one or more biopsy samples may comprise determiningspatially resolved information regarding the one or more biopsy samplesand/or the tissue; and

the one or more operational parameters may be calibrated, optimised orvaried on the basis of the spatially resolved information.

The one or more operational parameters may be calibrated, optimised orvaried depending on the position of the first device during thediagnostic or surgical procedure.

Analysing the one or more biopsy samples may comprise determining one ormore tissue types of the one or more biopsy samples and/or the tissue;and

the one or more operational parameters may be calibrated, optimised orvaried on the basis of the determined tissue types.

The one or more operational parameters may be calibrated, optimised orvaried depending on the type of tissue being analysed by the firstdevice during the diagnostic or surgical procedure.

The one or more tissue types may be selected from the group consistingof: (i) healthy tissue; (ii) diseased or tumour tissue; (iii) tissuecomprising both healthy and diseased cells, wherein the diseased cellsare optionally cancer cells; (iv) a type or grade of diseased or tumourtissue; (v) tissue at a border region of an organ and/or tumour; and/or(vi) tissue away from a border region of an organ and/or tumour.

The one or more operational parameters of the first device may comprise:(i) the magnitude and/or frequency of a voltage provided to the firstdevice; (ii) a temperature of the first device; (iii) the composition ofa matrix added to aerosol, smoke or vapour generated by the firstdevice; (iv) the temperature of a collision surface onto which aerosol,smoke or vapour generated by the first device is impacted; (v) a voltageapplied to a collision surface onto which aerosol, smoke or vapourgenerated by the first device is impacted; (vi) one or more operationalparameters of a mass analyser or filter for mass analysing analyte ionsand/or ions derived from the analyte ions; (vii) one or more operationalparameters of an ion mobility device for determining the ion mobility,collision cross section or interaction cross section of analyte ionsand/or ions derived from the analyte ions; and/or (viii) one or moreoperational parameters of a collision, reaction or fragmentation devicefor fragmenting or reacting analyte ions.

The method may comprise generating or updating a library or database onthe basis of the analysis, wherein the library or database is used forcalibrating or optimising the first device during the diagnostic orsurgical procedure.

According to an aspect there is provided apparatus comprising:

a first device for performing a diagnostic or surgical procedure,wherein the first device may be arranged and adapted to generate analyteions from tissue and to analyse the analyte ions; and

a control system arranged and adapted to calibrate, optimise or vary oneor more operational parameters of the first device for or during adiagnostic or surgical procedure on the basis of analysis of one or morebiopsy samples sampled from the tissue.

One or more of the one or more biopsy samples may comprise a sample oftissue having a longitudinal length.

The composition of the sample of tissue may vary or change along thelongitudinal length.

The longitudinal length may correspond to the depth within the tissue.

One or more of the one or more biopsy samples may comprise a biopsy coreor cylinder.

The analysis of the one or more biopsy samples may comprise analysis ofthe one or more biopsy samples performed using the first device.

The first device may comprise an ambient ionisation ion source.

The ambient ionisation ion source may comprise an ion source selectedfrom the group consisting of: (i) a rapid evaporative ionisation massspectrometry (“REIMS”) ion source; (ii) a desorption electrosprayionisation (“DESI”) ion source; (iii) a laser desorption ionisation(“LDI”) ion source; (iv) a thermal desorption ion source; (v) a laserdiode thermal desorption (“LDTD”) ion source; (vi) a desorptionelectro-flow focusing (“DEFFI”) ion source; (vii) a dielectric barrierdischarge (“DBD”) plasma ion source; (viii) an Atmospheric SolidsAnalysis Probe (“ASAP”) ion source; (ix) an ultrasonic assisted sprayionisation ion source; (x) an easy ambient sonic-spray ionisation(“EAST”) ion source; (xi) a desorption atmospheric pressurephotoionisation (“DAPPI”) ion source; (xii) a paperspray (“PS”) ionsource; (xiii) a jet desorption ionisation (“JeDI”) ion source; (xiv) atouch spray (“TS”) ion source; (xv) a nano-DESI ion source; (xvi) alaser ablation electrospray (“LAESI”) ion source; (xvii) a directanalysis in real time (“DART”) ion source; (xviii) a probe electrosprayionisation (“PEST”) ion source; (xix) a solid-probe assistedelectrospray ionisation (“SPA-EST”) ion source; (xx) a cavitronultrasonic surgical aspirator (“CUSA”) device; (xxi) a focussed orunfocussed ultrasonic ablation device; (xxii) a microwave resonancedevice; and (xxiii) a pulsed plasma RF dissection device.

The first device may be arranged and adapted to generate aerosol, smokeor vapour from the tissue, and to ionise the aerosol, smoke or vapour inorder to generate the analyte ions.

The apparatus may comprise a collision surface, wherein the apparatusmay be arranged and adapted to cause at least some of the aerosol, smokeor vapour to impact upon the collision surface in order to generate theanalyte ions.

The first device may comprise one or more electrodes arranged andadapted to contact the tissue in order to generate the aerosol, smoke orvapour.

The one or more electrodes may comprise a bipolar device or a monopolardevice.

The one or more electrodes may comprise either: (i) a monopolar device,wherein the apparatus optionally further comprises a separate returnelectrode; (ii) a bipolar device; or (iii) a multi-phase RF device,wherein the apparatus optionally further comprises a separate returnelectrode or electrodes.

The apparatus may comprise a device arranged and adapted to apply an ACor RF voltage to the one or more electrodes in order to generate theaerosol, smoke or vapour.

The device for applying the AC or RF voltage to the one or moreelectrodes may be arranged and adapted to apply one or more pulses ofthe AC or RF voltage to the one or more electrodes.

Application of the AC or RF voltage to the one or more electrodes maycause heat to be dissipated into the sample.

The first device and/or the one or more electrodes may comprise a rapidevaporation ionisation mass spectrometry (“REIMS”) device.

The first device may comprise a laser for irradiating the sample.

The apparatus may comprise: (i) a mass analyser or filter and/or ionmobility analyser for mass analysing and/or ion mobility analysinganalyte ions and/or ions derived from the analyte ions; (ii) an ionmobility device for determining the ion mobility, collision crosssection or interaction cross section of analyte ions and/or ions derivedfrom the analyte ions; and/or (iii) a fragmentation, reaction orcollision device for fragmenting or reacting analyte ions.

The first device may comprise an electrosurgical tool.

The first device may comprise an electrosurgical device, a diathermydevice, an ultrasonic device, hybrid ultrasonic electrosurgical device,surgical water jet device, hybrid electrosurgery, argon plasmacoagulation device, hybrid argon plasma coagulation device and water jetdevice and/or a laser device.

The control system may be arranged and adapted to calibrate, optimise orvary the one or more operational parameters on the basis of spatiallyresolved information determined from the analysis of the one or morebiopsy samples.

The control system may be arranged and adapted to calibrate, optimise orvary the one or more operational parameters depending on the position ofthe first device during the diagnostic or surgical procedure.

The control system may be arranged and adapted to calibrate, optimise orvary the one or more operational parameters on the basis of one or moretissue types determined from the analysis of the one or more biopsysamples.

The control system may be arranged and adapted to calibrate, optimise orvary the one or more operational parameters depending on the type oftissue being analysed by the first device during the diagnostic orsurgical procedure.

The one or more tissue types may be selected from the group consistingof: (i) healthy tissue; (ii) diseased or tumour tissue; (iii) tissuecomprising both healthy and diseased cells, wherein the diseased cellsare optionally cancer cells; (iv) a type or grade of diseased or tumourtissue; (v) tissue at a border region of an organ and/or tumour; and/or(vi) tissue away from a border region of an organ and/or tumour.

The one or more operational parameters of the first device may comprise:(i) the magnitude and/or frequency of a voltage provided to the firstdevice; (ii) a temperature of the first device; (iii) the composition ofa matrix added to aerosol, smoke or vapour generated by the firstdevice; (iv) the temperature of a collision surface onto which aerosol,smoke or vapour generated by the first device is impacted; (v) a voltageapplied to a collision surface onto which aerosol, smoke or vapourgenerated by the first device is impacted; (vi) one or more operationalparameters of a mass analyser or filter for mass analysing analyte ionsand/or ions derived from the analyte ions; (vii) one or more operationalparameters of an ion mobility device for determining the ion mobility,collision cross section or interaction cross section of analyte ionsand/or ions derived from the analyte ions; and/or (viii) one or moreoperational parameters of a collision, reaction or fragmentation devicefor fragmenting or reacting analyte ions.

The control system may be arranged and adapted to generate or update alibrary or database on the basis of the analysis, and to calibrate oroptimise the first device using the library or database during thediagnostic or surgical procedure.

In any of the various aspects and embodiments described herein, analysisof the analyte ions may result in spectrometric data and/or ion mobilitydata which may then be analysed.

Analysing the spectrometric data and/or ion mobility data may compriseanalysing one or more sample spectra so as to classify a sample.

Analysing the one or more sample spectra so as to classify the samplemay comprise unsupervised analysis of the one or more sample spectra(e.g., for dimensionality reduction) and/or supervised analysis of theone or more sample spectra (e.g., for classification)

Analysing the one or more sample spectra may comprise unsupervisedanalysis (e.g., for dimensionality reduction) followed by supervisedanalysis (e.g., for classification).

Analysing the one or more sample spectra may comprise using one or moreof: (i) univariate analysis; (ii) multivariate analysis; (iii) principalcomponent analysis (PCA); (iv) linear discriminant analysis (LDA); (v)maximum margin criteria (MMC); (vi) library-based analysis; (vii) softindependent modelling of class analogy (SIMCA); (viii) factor analysis(FA); (ix) recursive partitioning (decision trees); (x) random forests;(xi) independent component analysis (ICA); (xii) partial least squaresdiscriminant analysis (PLS-DA); (xiii) orthogonal (partial leastsquares) projections to latent structures (OPLS); (xiv) OPLSdiscriminant analysis (OPLS-DA); (xv) support vector machines (SVM);(xvi) (artificial) neural networks; (xvii) multilayer perceptron;(xviii) radial basis function (RBF) networks; (xix) Bayesian analysis;(xx) cluster analysis; (xxi) a kernelized method; and (xxii) subspacediscriminant analysis; (xxiii) k-nearest neighbours (KNN); (xxiv)quadratic discriminant analysis (QDA); (xxv) probabilistic principalcomponent Analysis (PPCA); (xxvi) non negative matrix factorisation;(xxvii) k-means factorisation; (xxviii) fuzzy c-means factorisation; and(xxix) discriminant analysis (DA).

Analysing the one or more sample spectra so as to classify the samplemay comprise developing a classification model or library using one ormore reference sample spectra.

Analysing the one or more sample spectra so as to classify the samplemay comprise performing linear discriminant analysis (LDA) (e.g., forclassification) after performing principal component analysis (PCA)(e.g., for dimensionality reduction).

Analysing the one or more sample spectra so as to classify the samplemay comprise performing a maximum margin criteria (MMC) process (e.g.,for classification) after performing principal component analysis (PCA)(e.g., for dimensionality reduction).

Analysing the one or more sample spectra so as to classify the samplemay comprise defining one or more classes within a classification modelor library.

Analysing the one or more sample spectra so as to classify the samplemay comprise defining one or more classes within a classification modelor library manually or automatically according to one or more class orcluster criteria.

The one or more class or cluster criteria for each class may be based onone or more of: a distance between one or more pairs of reference pointsfor reference sample spectra within a model space; a variance valuebetween groups of reference points for reference sample spectra within amodel space; and a variance value within a group of reference points forreference sample spectra within a model space.

The one or more classes may each be defined by one or more classdefinitions.

The one or more class definitions may comprise one or more of: a set ofone or more reference points for reference sample spectra, values,boundaries, lines, planes, hyperplanes, variances, volumes, Voronoicells, and/or positions, within a model space; and one or more positionswithin a class hierarchy.

Analysing the one or more sample spectra so as to classify the samplemay comprise using a classification model or library to classify one ormore unknown sample spectra.

Analysing the one or more sample spectra so as to classify the samplemay comprise classifying one or more sample spectra manually orautomatically according to one or more classification criteria.

The one or more classification criteria may comprise one or more of:

a distance between one or more projected sample points for one or moresample spectra within a model space and a set of one or more referencepoints for one or more reference sample spectra, values, boundaries,lines, planes, hyperplanes, volumes, Voronoi cells, or positions, withinthe model space being below a distance threshold or being the lowestsuch distance;

a position for one or more projected sample points for one or moresample spectra within a model space being one side or other of one ormore reference points for one or more reference sample spectra, values,boundaries, lines, planes, hyperplanes, or positions, within the modelspace;

a position for one or more projected sample points for one or moresample spectra within a model space being within one or more volumes orVoronoi cells within the model space; and

a probability or classification score being above a probability orclassification score threshold or being the highest such probability orclassification score.

According to an aspect there is provided a mass and/or ion mobilityanalyser comprising apparatus as described above.

According to an aspect there is provided a method of mass spectrometryand/or method of ion mobility spectrometry comprising a method asdescribed above.

The mass and/or ion mobility spectrometer may obtain data in negativeion mode only, positive ion mode only, or in both positive and negativeion modes. Positive ion mode spectrometric data may be combined orconcatenated with negative ion mode spectrometric data.

Ion mobility spectrometric data may be obtained using different ionmobility drift gases and/or dopants. This data may then be combined orconcatenated.

Various embodiments are contemplated which relate to generating smoke,aerosol or vapour from a target (details of which are provided elsewhereherein) using an ambient ionisation ion source. The aerosol, smoke orvapour may then be mixed with a matrix and aspirated into a vacuumchamber of a mass spectrometer and/or ion mobility spectrometer. Themixture may be caused to impact upon a collision surface causing theaerosol, smoke or vapour to be ionised by impact ionization whichresults in the generation of analyte ions. The resulting analyte ions(or fragment or product ions derived from the analyte ions) may then bemass analysed and/or ion mobility analysed and the resulting massspectrometric data and/or ion mobility spectrometric data may besubjected to multivariate analysis or other mathematical treatment inorder to determine one or more properties of the target in real time.

According to an embodiment the first device for generating aerosol,smoke or vapour from the target may comprise a tool which utilises an RFvoltage, such as a continuous RF waveform.

Other embodiments are contemplated wherein the first device forgenerating aerosol, smoke or vapour from the target may comprise anargon plasma coagulation (“APC”) device. An argon plasma coagulationdevice involves the use of a jet of ionised argon gas (plasma) that isdirected through a probe. The probe may be passed through an endoscope.Argon plasma coagulation is essentially a non-contact process as theprobe is placed at some distance from the target. Argon gas is emittedfrom the probe and is then ionized by a high voltage discharge (e.g., 6kV). High-frequency electric current is then conducted through the jetof gas, resulting in coagulation of the target on the other end of thejet. The depth of coagulation is usually only a few millimetres.

The first device, surgical or electrosurgical tool, device or probe orother sampling device or probe disclosed in any of the aspects orembodiments herein may comprise a non-contact surgical device, such asone or more of a hydrosurgical device, a surgical water jet device, anargon plasma coagulation device, a hybrid argon plasma coagulationdevice, a water jet device and a laser device.

A non-contact surgical device may be defined as a surgical devicearranged and adapted to dissect, fragment, liquefy, aspirate, fulgurateor otherwise disrupt biologic tissue without physically contacting thetissue. Examples include laser devices, hydrosurgical devices, argonplasma coagulation devices and hybrid argon plasma coagulation devices.

As the non-contact device may not make physical contact with the tissue,the procedure may be seen as relatively safe and can be used to treatdelicate tissue having low intracellular bonds, such as skin or fat.

According to various embodiments the mass spectrometer and/or ionmobility spectrometer may obtain data in negative ion mode only,positive ion mode only, or in both positive and negative ion modes.Positive ion mode spectrometric data may be combined or concatanatedwith negative ion mode spectrometric data. Negative ion mode can provideparticularly useful spectra for classifying aerosol, smoke or vapoursamples, such as aerosol, smoke or vapour samples from targetscomprising lipids.

Ion mobility spectrometric data may be obtained using different ionmobility drift gases, or dopants may be added to the drift gas to inducea change in drift time of one or more species. This data may then becombined or concatenated.

It will be apparent that the requirement to add a matrix or a reagentdirectly to a sample may prevent the ability to perform in vivo analysisof tissue and also, more generally, prevents the ability to provide arapid simple analysis of target material.

According to other embodiments the ambient ionisation ion source maycomprise an ultrasonic ablation ion source or a hybridelectrosurgical-ultrasonic ablation source that generates a liquidsample which is then aspirated as an aerosol. The ultrasonic ablationion source may comprise a focused or unfocussed ultrasound.

Optionally, the first device comprises or forms part of an ion sourceselected from the group consisting of: (i) a rapid evaporativeionisation mass spectrometry (“REIMS”) ion source; (ii) a desorptionelectrospray ionisation (“DESI”) ion source; (iii) a laser desorptionionisation

(“LDI”) ion source; (iv) a thermal desorption ion source; (v) a laserdiode thermal desorption (“LDTD”) ion source; (vi) a desorptionelectro-flow focusing (“DEFFI”) ion source; (vii) a dielectric barrierdischarge (“DBD”) plasma ion source; (viii) an Atmospheric SolidsAnalysis Probe (“ASAP”) ion source; (ix) an ultrasonic assisted sprayionisation ion source; (x) an easy ambient sonic-spray ionisation(“EAST”) ion source; (xi) a desorption atmospheric pressurephotoionisation (“DAPPI”) ion source; (xii) a paperspray (“PS”) ionsource; (xiii) a jet desorption ionisation (“JeDI”) ion source; (xiv) atouch spray (“TS”) ion source; (xv) a nano-DESI ion source; (xvi) alaser ablation electrospray (“LAESI”) ion source; (xvii) a directanalysis in real time (“DART”) ion source; (xviii) a probe electrosprayionisation (“PESI”) ion source; (xix) a solid-probe assistedelectrospray ionisation (“SPA-ESI”) ion source; (xx) a cavitronultrasonic surgical aspirator (“CUSA”) device; (xxi) a hybridCUSA-diathermy device; (xxii) a focussed or unfocussed ultrasonicablation device; (xxiii) a hybrid focussed or unfocussed ultrasonicablation and diathermy device; (xxiv) a microwave resonance device;(xxv) a pulsed plasma RF dissection device; (xxvi) an argon plasmacoagulation device; (xxvi) a hybrid pulsed plasma RF dissection andargon plasma coagulation device; (xxvii) a hybrid pulsed plasma RFdissection and JeDI device; (xxviii) a surgical water/saline jet device;(xxix) a hybrid electrosurgery and argon plasma coagulation device; and(xxx) a hybrid argon plasma coagulation and water/saline jet device.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments will now be described, by way of example only, andwith reference to the accompanying drawings in which:

FIG. 1 shows schematically a variety of microbes that are present in thehuman microbiome;

FIG. 2 shows schematically various mucosa or mucosal membranes which arepresent in the human body;

FIG. 3 shows schematically a mucosa or mucosal membrane comprisingbiological tissue and bacteria;

FIG. 4 shows schematically how analytes present in a mucosa may beuseful in identifying a number of clinical disorders;

FIG. 5 shows schematically how metabolomic profiling of analytes from amucosal membrane can be useful in identifying clinical disorders such asallergies, inflammation and pre-term delivery;

FIG. 6 shows various approaches for microbial analysis together with areal time rapid and direct analysis method using ambient massspectrometry according to various embodiments;

FIG. 7 illustrates the technique of Desorption Electrospray Ionisation(“DESI”) according to various embodiments;

FIGS. 8A-C show schematically a desorption electrospray ionisation(“DESI”) mass spectrometry setup for swab analysis according to variousembodiments;

FIG. 9 shows schematically mucosal membrane sampling from selected partsof the human body (e.g., urogenital tract, oral or nose cavity) usingmedical cotton swabs as a sampling device wherein the surface of themedical swab may then be directly analysed by desorption electrosprayionisation (“DESI”) mass spectrometry without prior sample preparationprocedures according to various embodiments;

FIG. 10A shows averaged negative-ion desorption electrospray ionisation(“DESI”) mass spectra from vaginal, oral and nasal mucosa recorded usinga Xevo G2-S Q-Tof ® mass spectrometer and FIG. 10B shows a PCA and MMCscore plot for vaginal (n=68), oral (n=15) and nasal (n=20) mucosaacquired with desorption electrospray ionisation (“DESI”) massspectrometry;

FIG. 11 shows desorption electrospray ionisation (“DESI”) massspectrometry spectra of vaginal, oral and nasal mucosal membranes in anegative ion mode obtained from medical cotton swabs, together withprincipal component analysis (PCA) and maximum margin criterion analysisproviding a separation between different mucosal classes (nasal, oral,vaginal) with a prediction accuracy ranging from 92-100% obtained byleave one out cross validation;

FIG. 12 shows a desorption electrospray ionisation (“DESI”) massspectrum of pregnant vaginal mucosal membrane obtained in negative ionmode from a medical cotton swab, wherein the urogenital mucosa was foundto produce cholesterol sulphate [M−H]⁻ having a mass to charge ratio of465.41 as the most abundant lipid species as well as a differentglycerophosholipids species such as glycerophosphoethanolamine (PE)[PE(40:7)-H]⁻ having a mass to charge ratio of 788.50,glycerophosphoserine (PS) [PS(34:1)-H]⁻ having a mass to charge ratio of760.50 and glycerophosphoinositol (PI) [PI(36:1)-H]⁻ having a mass tocharge ratio of 863.58;

FIG. 13A shows averaged desorption electrospray ionisation (“DESI”) massspectra from a pregnant (highlighted in blue) and a non-pregnant group(highlighted in red) acquired in negative ion mode in the mass range m/z150-1000, FIG. 13B shows principal component analysis and discriminatoryanalysis using recursive maximum margin criterion (“RMMC”), FIG. 13Cshows analysis with leave-one-out cross-validation for enhancedseparation of group classes with highly accurate identification (>80%)based on chemical signatures in the vaginal mucosal membrane, FIG. 13Dshows box plots indicating significant differences of the abundance forselected peaks between non-pregnant and pregnant vaginal mucosalmembranes mainly in the mass to charge ratio (“m/z”) range 550-1000 andFIG. 13E shows the leave-one-out cross-validation;

FIG. 14A shows desorption electrospray ionisation (“DESI”) mass spectralanalysis of a bacteria sample on a swab in accordance with variousembodiments and shows that bacterial samples can be detected using DESIand FIG. 14B shows a comparison with rapid evaporative ionisation massspectrometry (“REIMS”) analysis in conjunction with a Time of Flightmass analysis of a bacterial sample directly from an agar plate;

FIG. 15A shows averaged desorption electrospray ionisation (“DESI”) massspectra of diverse analysed microorganism species including Candidaalbicans, Pseudomonas montelli, Staphylococcus epidermis, Moraxellacatarrhalis, Klebsiella pneumonia and Lactobacillus sp as well aspregnant vaginal mucosa, FIG. 15B shows a PCA plot showing a separationbetween the vaginal mucosa (pregnant and non-pregnant group) from themicroorganism species within the first two components and FIG. 15C showsa separation between the different bacteria and fungi species;

FIG. 16 shows schematically desorption electrospray ionisation (“DESI”)mass spectrometry analysis, rapid evaporative ionisation massspectrometry (“REIMS”) mass spectrometry analysis and culturing basedanalysis of a sample on a swab according to various embodiments;

FIG. 17A shows desorption electrospray ionisation (“DESI”) mass spectraldata wherein a swab may be continuously rotated when subjected todesorption electrospray ionisation (“DESI”) ionisation in order toimprove the signal intensity and FIG. 17B shows rapid evaporativeionisation mass spectrometry (“REIMS”) mass spectral data wherein a swabmay be dipped, soaked or otherwise immersed in a fluid (such as water)prior to be being subjected to rapid evaporative ionisation massspectrometry (“REIMS”) analysis in order to improve the signalintensity;

FIG. 18 shows schematically the rapid evaporative ionisation massspectrometry (“REIMS”) technique in accordance with various embodiments;

FIG. 19 illustrates various advantages and disadvantages associated withstandard cotton swabs and coated or chemically modified swabs accordingto various embodiments;

FIG. 20A illustrates a mass spectrum obtained when using a standardcotton swab, FIG. 20B shows how an improved sensitivity (especiallyimproved lipid signal) may be obtained using a modified swab and FIG.20C shows how an improved sensitivity (especially improved lipid signal)may be obtained using a modified swab according to various embodiments;

FIG. 21 shows various solid-phase microextraction (“SPME”) coatedmaterials for mucosal membrane sampling in accordance with variousembodiments;

FIG. 22 shows schematically a solid-phase microextraction (“SPME”) swabsample preparation workflow for the extraction of analytes in salivamatrix followed by desorption electrospray ionisation (“DESI”) massspectrometry analysis in accordance with various embodiments;

FIG. 23A shows a saliva mass spectrum obtained in negative ion mode(left) and positive ion mode (right) using a standard medical swab, FIG.23B shows a saliva mass spectrum obtained in negative ion mode (left)and positive ion mode (right) using a standard medical swab coated withthree layers of C18 (octadecyl) sorbent, FIG. 23C shows a saliva massspectrum obtained in negative ion mode (left) and positive ion mode(right) using a standard medical swab coated with three layers of C18(octadecyl) EC (end capped) sorbent, FIG. 23D shows a saliva massspectrum obtained in negative ion mode (left) and positive ion mode(right) using a standard medical swab coated with three layers of HLB(hydrophilic-lipophilic-balanced) sorbent and FIG. 23E shows a salivamass spectrum obtained in negative ion mode (left) and positive ion mode(right) using a standard medical swab coated with three layers of DVB(divinyl benzene) WAX (weak anion exchange) sorbent;

FIG. 24 shows a spectrum observed when analysing stool samples usingrapid evaporative ionisation mass spectrometry (“REIMS”) analysis;

FIG. 25A shows a Desorption Electrospray Ionisation (“DESI”) device,FIG. 25B shows a graph of intensity versus inlet capillary temperaturefor analysis of fatty acids using a Waters Synapt® mass spectrometer,FIG. 25C shows a graph of intensity versus inlet capillary temperaturefor analysis of fatty acids using a Waters Xevo® mass spectrometer, FIG.25D shows a graph of intensity versus inlet capillary temperature foranalysis of phospholipids using a Waters Synapt® mass spectrometer andFIG. 25E shows a graph of intensity versus inlet capillary temperaturefor analysis of phospholipids using a Waters Xevo® mass spectrometer;

FIG. 26 shows a needle biopsy procedure in which a biopsy needle is usedto extract a biopsy core from a patient;

FIG. 27 shows a biopsy needle comprising a rapid evaporative ionisationmass spectrometry (“REIMS”) electrode in accordance with an embodiment;

FIG. 28 shows a method of analysis that comprises building aclassification model according to various embodiments;

FIG. 29 shows a set of reference sample spectra obtained from twoclasses of known reference samples;

FIG. 30 shows a multivariate space having three dimensions defined byintensity axes, wherein the multivariate space comprises pluralreference points, each reference point corresponding to a set of threepeak intensity values derived from a reference sample spectrum;

FIG. 31 shows a general relationship between cumulative variance andnumber of components of a PCA model;

FIG. 32 shows a PCA space having two dimensions defined by principalcomponent axes, wherein the PCA space comprises plural transformedreference points or scores, each transformed reference point or scorecorresponding to a reference point of FIG. 30;

FIG. 33 shows a PCA-LDA space having a single dimension or axis, whereinthe LDA is performed based on the PCA space of FIG. 32, the PCA-LDAspace comprising plural further transformed reference points or classscores, each further transformed reference point or class scorecorresponding to a transformed reference point or score of FIG. 32;

FIG. 34 shows a method of analysis that comprises using a classificationmodel according to various embodiments;

FIG. 35 shows a sample spectrum obtained from an unknown sample;

FIG. 36 shows the PCA-LDA space of FIG. 33, wherein the PCA-LDA spacefurther comprises a PCA-LDA projected sample point derived from the peakintensity values of the sample spectrum of FIG. 35;

FIG. 37 shows a method of analysis that comprises building aclassification library according to various embodiments; and

FIG. 38 shows a method of analysis that comprises using a classificationlibrary according to various embodiments.

DETAILED DESCRIPTION

Various embodiments will now be described in more detail. Some of theembodiments which are described in more detail relate to using aDesorption Electrospray Ionisation (“DESI”) ion source to analyse astandard medical swab.

However, other embodiments are contemplated wherein a different ambientionisation ion source may be used.

Ambient Ionisation Ion Sources

Various embodiments as described herein are described in the context ofusing a Desorption Electrospray Ionisation (“DESI”) ion source togenerate a spray of electrically charged droplets. However, otherembodiments are contemplated wherein other devices may be used togenerate analyte ions.

The devices or ion sources may comprise ambient ionisation ion sourceswhich are characterised by the ability to generate analyte ions from anative or unmodified target. By way of contrast, other types ofionisation ion sources such as Matrix Assisted Laser DesorptionIonisation (“MALDI”) ion sources require the addition of a matrix orreagent to the sample prior to ionisation.

It will be apparent that the requirement to add a matrix or a reagent toa sample prevents the ability to perform in vivo analysis of tissue andalso, more generally, prevents the ability to provide a rapid simpleanalysis of target material.

In contrast, therefore, ambient ionisation techniques are particularlybeneficial since firstly they do not require the addition of a matrix ora reagent (and hence are suitable for the analysis of in vivo tissue)and since secondly they enable a rapid simple analysis of targetmaterial to be performed.

A number of different ambient ionisation techniques are known and areintended to fall within the scope of the present invention. DesorptionElectrospray Ionisation (“DESI”) was the first ambient ionisationtechnique to be developed and was disclosed in 2004. Since 2004, anumber of other ambient ionisation techniques have been developed. Theseambient ionisation techniques differ in their precise ionisation methodbut they share the same general capability of generating gas-phase ionsdirectly from native (i.e. untreated or unmodified) samples. Aparticular benefit of the various ambient ionisation techniques whichfall within the scope of the present invention is that the variousambient ionisation techniques do not require any prior samplepreparation. As a result, the various ambient ionisation techniquesenable both in vivo and ex vivo tissue samples to be analysed withoutnecessitating the time and expense of adding a matrix or reagent to thetissue sample or other target material.

A list of ambient ionisation techniques which are intended to fallwithin the scope of the present invention are given in the followingtable:

Acronym Ionisation technique DESI Desorption electrospray ionisationDeSSI Desorption sonic spray ionisation DAPPI Desorption atmosphericpressure photoionisation EASI Easy ambient sonic-spray ionisation JeDIJet desorption electrospray ionisation TM-DESI Transmission modedesorption electrospray ionisation LMJ-SSP Liquid microjunction-surfacesampling probe DICE Desorption ionisation by charge exchange Nano-DESINanospray desorption electrospray ionisation EADESI Electrode-assisteddesorption electrospray ionisation APTDCI Atmospheric pressure thermaldesorption chemical ionisation V-EASI Venturi easy ambient sonic-sprayionisation AFAI Air flow-assisted ionisation LESA Liquid extractionsurface analysis PTC-ESI Pipette tip column electrospray ionisationAFADESI Air flow-assisted desorption electrospray ionisation DEFFIDesorption electro-flow focusing ionisation ESTASI Electrostatic sprayionisation PASIT Plasma-based ambient sampling ionisation transmissionDAPCI Desorption atmospheric pressure chemical ionisation DART Directanalysis in real time ASAP Atmospheric pressure solid analysis probeAPTDI Atmospheric pressure thermal desorption ionisation PADI Plasmaassisted desorption ionisation DBDI Dielectric barrier dischargeionisation FAPA Flowing atmospheric pressure afterglow HAPGDI Heliumatmospheric pressure glow discharge ionisation APGDDI Atmosphericpressure glow discharge desorption ionisation LTP Low temperature plasmaLS-APGD Liquid sampling-atmospheric pressure glow discharge MIPDIMicrowave induced plasma desorption ionisation MFGDP Microfabricatedglow discharge plasma RoPPI Robotic plasma probe ionisation PLASI Plasmaspray ionisation MALDESI Matrix assisted laser desorption electrosprayionisation ELDI Electrospray laser desorption ionisation LDTD Laserdiode thermal desorption LAESI Laser ablation electrospray ionisationCALDI Charge assisted laser desorption ionisation LA-FAPA Laser ablationflowing atmospheric pressure afterglow LADESI Laser assisted desorptionelectrospray ionisation LDESI Laser desorption electrospray ionisationLEMS Laser electrospray mass spectrometry LSI Laser spray ionisationIR-LAMICI Infrared laser ablation metastable induced chemical ionisationLDSPI Laser desorption spray post-ionisation PAMLDI Plasma assistedmultiwavelength laser desorption ionisation HALDI High voltage-assistedlaser desorption ionisation PALDI Plasma assisted laser desorptionionisation ESSI Extractive electrospray ionisation PESI Probeelectrospray ionisation ND-ESSI Neutral desorption extractiveelectrospray ionisation PS Paper spray DIP-APCI Direct inletprobe-atmospheric pressure chemical ionisation TS Touch spray Wooden-tipWooden-tip electrospray CBS-SPME Coated blade spray solid phasemicroextraction TSI Tissue spray ionisation RADIO Radiofrequencyacoustic desorption ionisation LIAD-ESI Laser induced acousticdesorption electrospray ionisation SAWN Surface acoustic wavenebulization UASI Ultrasonication-assisted spray ionisation SPA-nanoESISolid probe assisted nanoelectrospray ionisation PAUSI Paper assistedultrasonic spray ionisation DPESI Direct probe electrospray ionisationESA-Py Electrospray assisted pyrolysis ionisation APPIS Ambient pressurepyroelectric ion source RASTIR Remote analyte sampling transport andionisation relay SACI Surface activated chemical ionisation DEMIDesorption electrospray metastable-induced ionisation REIMS Rapidevaporative ionisation mass spectrometry SPAM Single particle aerosolmass spectrometry TDAMS Thermal desorption-based ambient massspectrometry MAII Matrix assisted inlet ionisation SAII Solvent assistedinlet ionisation SwiFERR Switched ferroelectric plasma ioniser LPTDLeidenfrost phenomenon assisted thermal desorption

According to an embodiment the ambient ionisation ion source maycomprise a rapid evaporative ionisation mass spectrometry (“REIMS”) ionsource wherein an RF voltage is applied to electrodes in order togenerate an aerosol or plume of surgical smoke by Joule heating.

However, it will be appreciated that numerous other ambient ion sourcesincluding those referred to above may be utilised. For example,according to another embodiment the ambient ionisation ion source maycomprise a laser ionisation ion source. According to an embodiment thelaser ionisation ion source may comprise a mid-IR laser ablation ionsource. For example, there are several lasers which emit radiation closeto or at 2.94 μm which corresponds with the peak in the water absorptionspectrum. According to various embodiments the ambient ionisation ionsource may comprise a laser ablation ion source having a wavelengthclose to 2.94 μm, i.e., on the basis of the high absorption coefficientof water at 2.94 μm. According to an embodiment the laser ablation ionsource may comprise an Er:YAG laser which emits radiation at 2.94 μm.

Other embodiments are contemplated wherein a mid-infrared opticalparametric oscillator (“OPO”) may be used to produce a laser ablationion source having a longer wavelength than 2.94 μm. For example, anEr:YAG pumped ZGP-OPO may be used to produce laser radiation having awavelength of e.g. 6.1 μm, 6.45 μm or 6.73 μm. In some situations it maybe advantageous to use a laser ablation ion source having a shorter orlonger wavelength than 2.94 μm since only the surface layers will beablated and less thermal damage may result. According to an embodiment aCo:MgF2 laser may be used as a laser ablation ion source wherein thelaser may be tuned from 1.75-2.5 μm. According to another embodiment anoptical parametric oscillator (“OPO”) system pumped by a Nd:YAG lasermay be used to produce a laser ablation ion source having a wavelengthbetween 2.9-3.1 μm. According to another embodiment a CO₂ laser having awavelength of 10.6 μm may be used to generate the aerosol, smoke orvapour.

According to other embodiments the ambient ionisation ion source maycomprise an ultrasonic ablation ion source which generates a liquidsample which is then aspirated as an aerosol. The ultrasonic ablationion source may comprise a focused or unfocussed source. According to anembodiment the first device for generating aerosol, smoke or vapour fromone or more regions of a target may comprise an electrosurgical toolwhich utilises a continuous RF waveform. According to other embodimentsa radiofrequency tissue dissection system may be used which is arrangedto supply pulsed plasma RF energy to a tool. The tool may comprise, forexample, a PlasmaBlade®. Pulsed plasma RF tools operate at lowertemperatures than conventional electrosurgical tools (e.g. 40-170° C.c.f. 200-350° C.) thereby reducing thermal injury depth. Pulsedwaveforms and duty cycles may be used for both cut and coagulation modesof operation by inducing electrical plasma along the cutting edge(s) ofa thin insulated electrode.

Real-time rapid analysis using desorption electrospray ionisation(“DESI”) mass spectrometry of medical swabs

According to various embodiments swabs, medical swabs or standardmedical swabs may be directly analysed using desorption electrosprayionisation (“DESI”) mass spectrometry, and in particular the specificmicrobe(s) on the surface of swabs may be identified by their chemicalsignature within a short period of time.

The rapid identification of specific microbes from the surface of swabsenables rapid diagnosis of various infections to be made. Furthermore,biomarkers, such as metabolomic, inflammatory and/or microbial markerscan be analysed, e.g., identified or determined, which e.g. enables therapid analysis, e.g., identification, of different diseases, such ascancer, dysbiosis, infections, and/or any of the other diseases listedelsewhere herein.

Various embodiments will be described in more detail below which relateto mucosal analysis, e.g., diagnostics.

One of many potential applications of the various techniques which aredisclosed herein is the ability to identify, by analysing vaginalmucosal samples, whether or not a patient is at an increased risk ofsuffering a preterm (premature) delivery. Results according to variousembodiments may optionally be compared with standard microbial testing.

A real-time rapid medical swab analysis approach is disclosed whichutilises desorption electrospray ionisation (“DESI”) mass spectrometryto reveal biomarkers such as pathogenic and/or inflammatory metabolomicmarkers.

In particular, various chemically modified swabs for use with desorptionelectrospray ionisation (“DESI”) are disclosed. Various chemicallymodified swabs have been found to exhibit an improved sensitivitycompared with conventional (non-modified) swabs.

It has also been found that a significant enhancement in signalintensity can be obtained by rotating or continuously rotating the swabswhilst analysing the swab using a desorption electrospray ionisation(“DESI”) ion source.

Further embodiments are also disclosed below which relate to methods ofrapid evaporative ionisation mass spectrometry (“REIMS”) analysis(rather than desorption electrospray ionisation (“DESI”) analysis) of aswab wherein the swab is dipped, soaked or otherwise immersed in a fluid(such as water) prior to be being subjected to rapid evaporativeionisation mass spectrometry (“REIMS”) analysis.

Soaking a swab in a fluid such as water prior to rapid evaporativeionisation mass spectrometry has been found to have the effect ofimproving the signal intensity.

According to various further embodiments, desorption electrosprayionisation (“DESI”) mass spectrometry of swabs may be used, e.g. fortoxicological screening, such as on-site emergency toxicologicalscreening, drug testing, such as roadside drug testing, doping testingand so on.

Desorption Electrospray Ionisation (“DESI”) Mass Spectrometry Analysisof Mucosal Samples

Various embodiments will now be described in more detail which relate toa non-invasive approach for mucosal analysis, e.g., diagnostics.

Medical swabs are a standard collection device for mucosal membranes andare commonly used for diagnosis of pathogenic related diseases. Routineclinical microbiology techniques for mucosal swab diagnostics are timeconsuming, lack sensitivity and are generally qualitative. Standardmedical swabs which have been used to sample a mucosal membrane are sentto a microbiological laboratory where the sample is then analysed byculturing microbes. However, this conventional approach typically takes24-48 hours and delays diagnosis of the patient.

By way of contrast, various embodiments will now be described in moredetail which allow mucosal membrane samples to be analysed immediatelyor in real time, thereby avoiding the 24-48 hour delay which is commonaccording to conventional techniques.

In particular, according to various embodiments, a method is providedthat comprises providing a biological sample on a swab, directing aspray of charged droplets onto a surface of the swab in order togenerate a plurality of analyte ions, and analysing the analyte ions.

Various embodiments relate to a method for rapid, direct analysis ofmedical swabs by desorption electrospray ionisation mass spectrometrywithout the need for extensive extraction protocols. According tovarious embodiments, ionisation of mucosal biomass occurs directly froma medical swab, such as a standard medical rayon swab, which may berotating, before analysis in a mass and/or ion mobility spectrometer,e.g. for online chemical monitoring. According to various embodiments,multivariate modelling of acquired mass spectral fingerprints permitsdiscrimination of differing mucosal surfaces, characterisation ofbiochemical alterations, e.g. induced by pregnancy, and/or rapididentification of intact bacterial and fungal species. The directmedical swab analysis by desorption electrospray ionisation massspectrometry according to various embodiments may be used in a widerange of clinical applications, including rapid mucosal diagnosticsand/or characterisation of clinically relevant changes in mucosalbiochemistry.

Various embodiments relate to a non-invasive and culture-independentmethod that allows profiling, e.g., metabolomic profiling of mucosalmembranes to be performed by direct analysis of clinical swabs usingdesorption electrospray ionisation (“DESI”), e.g. within a few minutes.These swabs may be used to give a fast diagnosis of disease including,e.g.: (i) microbial infection; (ii) dysbiosis; (iii) immunologicaldisorders; (iv) cancer; and/or any of the other diseases listedelsewhere herein.

As described further below, a total of n=85 mucosal membrane models werecollected from three cohorts (urogenital tract, nasal and oral cavity).The mucosal membrane samples were subjected to desorption electrosprayionisation (“DESI”) mass spectral analysis and the resulting massspectral data was subjected to multivariate statistical analysis.Multivariate statistical analysis was able to separate different mucosaclasses and biomarker changes that can be associated with a diversemicrobiome within the mucosa.

The microbiome comprises the community of microorganisms that inhabithuman or non-human animal bodies, e.g., human bodies. Humans andnon-human animals have co-evolved with microbes as a symbiotic system.Complex reactions of microbe communities influence health and disease.

FIG. 1 illustrates a variety of microbes that may be present in thehuman microbiome. As shown in FIG. 1, the human microbiome may includevarious bacteria, fungi, archaea, viruses, yeasts, protozoa, etc. whichmay be present, e.g., in the mouth, pharynx, respiratory system, skin,stomach, intestines, and/or urogenital tract, etc.

A “microbe”, also known as a micro-organism, is an organism which is toosmall to be visible to the naked eye, i.e. is microscopic. A microbe maybe selected from bacteria, fungi, archaea, algae, protozoa and viruses.Although the terms bacteria, fungi, archaea, algae, protozoa and virusestechnically denote the plural form, it is common practice to use themalso to denote the singular form. Consequently, the terms “bacteria” and“bacterium” are used interchangeably herein; the terms “fungi” and“fungus” are used interchangeably herein; the terms “archaea” and“archaeum” are used interchangeably herein; the terms “protozoa” and“protozoum” are used interchangeably herein; and the terms “viruses” and“virus” are used interchangeably herein.

In the case of a microbe, analysis may optionally be on any taxonomiclevel, for example, at the Kingdom, Phylum or Division, Class, Order,Family, Genus, Species and/or Strain level.

“Taxonomy” is the classification of organisms, and each level ofclassification may be referred to as a “taxon” (plural: taxa). Organismsmay be classified into the following taxa in increasing order ofspecificity: Kingdom, Phylum or Division, Class, Order, Family, Genus,Species and Strain. Further subdivisions of each taxon may exist. Itmust be appreciated that within the vast scientific community there aresome discrepancies within some taxonomic classifications. There may alsobe a lack of consensus with regard to the nomenclature of certainmicrobes, resulting in a particular microbe having more than one name orin two different microbes having the same name.

As a shorthand, the term “type” of microbe is used to refer to a microbethat differs from another microbe at any taxonomic level.

In some embodiments, the microbe may be selected from bacteria, fungi,archaea, algae and protozoa. In some embodiments, it may be selectedfrom bacteria and fungi. In some embodiments, it may be selected frombacteria.

The microbe may be single-cellular or multi-cellular. If the microbe isa fungus, it may optionally be filamentous or single-cellular, e.g., ayeast.

A fungus may optionally be yeast. It may optionally be selected from thegenus Aspergillus, Arthroascus, Brettanomyces Candida, Cryptococcus,Debaryomyces, Geotrichum, Pichia, Rhodotorula, Saccharomyces,Trichosporon and Zygotorulaspora.

It may optionally be selected from the species Arthroascus schoenii,Brettanomyces bruxellensis, Candida albicans, C. ascalaphidarum,C.amphixiae, C.antarctica, C.argentea, C. atlantica, C. atmosphaerica,C. blattae, C. bromeliacearum, C. carpophila, C. carvajalis, C.cerambycidarum, C. chauliodes, C. corydali, C. dosseyi, C. dubliniensis,C. ergatensis, Cfructus, C.glabrata, C.fermentati, C.guilliermondii,C.haemulonii, C.insectamens, C.insectorum, C.intermedia, Cjeffresii,C.kefyr, C.keroseneae, C.krusei, C.lusitaniae, C.lyxosophila, C.maltosa,C. marina, C.membranifaciens, C.milleri, C.mogii, C.oleophila,C.oregonensis, C.parapsilosis, C. quercitrusa, C. rugosa, C. sake, C.shehatea, C. temnochilae, C. tenuis, C. theae, C. tolerans, C.tropicalis, C. tsuchiyae, C. sinolaborantium, C. sojae, C. subhashii,C.viswanathii, C.utilis, C.ubatubensis, C.zemplinina, Cryptococcusneoformans, Cryptococcus uniguttulatus, Debaryomyces carsonii,Geotrichum capitatum, Trichosporon asahii, Trichosporon mucoides,Trichosporon inkin, Saccharomyces cerevisiae, Pichia acaciae, Pichiaanomala, Pichia capsulata, Pichia farinosa, Pichia guilliermondii,Pichia spartinae, Pichia ohmeri, Rhodotorula glutinous, Rhodotorulamucilaginosa, Saccharomyces boulardii, Saccharomyces cerevisiae and/orZygotorulaspora florentinus.

The protozoa may optionally be selected from the group of amoebae,flagellates, ciliates or sporozoa. It may optionally be selected fromthe genus Acanthamoeba, Babesia, Balantidium, Cryptosporidium,Dientamoeba, Entamoeba, Giardia, Leishmania, Naegleria, PlasmodiumParamecium, Trichomonas, Trypanosoma, Typanosoma and Toxoplasma.

The protozoa may optionally be of the species Balantidium coli,Entamoeba histolytica, Giardia lamblia (also known as Giardiaintestinalis, or Giardia duodenalis), Leishmania donovani, L. tropica,L. brasiliensis, Plasmodium falciparum, P. vivax, P. ovale, P. malariae,P. knowlesi, P. reichenowi, P. gaboni, P. mexicanum, P. floridenseTrypanosoma brucei, Typanosoma evansi, Trypanosoma rhodesiense,Trypanosoma cruzi and Toxoplasma gondii.

The bacteria may optionally be selected from the phylum Aquficae,Thermotogae, Thermodesulfobacteria, Deinococcus-Thermus, Chrysiogenetes,Chloroflexi, Thermomicrobia, Nitrospira, Deferribacteres, Cyanobacteria,Chlorobi, Proteobacteria, Firmicutes, Actinobacteria, Planctomycetes,Chlamydiae, Spirochaetes, Fibrobacteres, Acidobacteria, Bacteroidetes,Fusobacteria, Verrucomicrobia, Dictyoglomi, Gemmatomonadetes and/orLentisphaerae.

The bacteria may optionally be selected from the class Actinobacteria,Alphaproteobacteria, Bacilli, Betaproteobacteria, Clostridia,Deltaproteobacteria, Epsilonproteobacteria, Flavobacteriaceae,Fusobacteria, Gammaproteobacteria, Mikeiasis, Mollicute, orNegativicutes.

The bacteria may optionally be of the Order Aeromonadales,Actinomycetales, Bacillales, Bacteroidales, Bifidobacteriales,Burkholderiales, Campylobacterales, Caulobacterales, Cardiobacteriales,Clostridiales, Enterobacteriales, Flavobacteriales, Fusobacteriales,Lactobacillales, Micrococcales, Neisseriales, Pasteurellales,Pseudomonadales, Rhizobiales, Rhodospirillales, Selenomonadales,Vibrionales and/or Xanthomonadales.

The bacteria may optionally be selected from the FamilyAcetobacteraceae, Alcaligenaceae, Bacillaceae, Bacteroidaceae,Burkholderiaceae, Caulobacteraceae, Comamonadaceae, Enterobacteriaceae,Flavobacteriaceae, Fusobacteriaceae Nocardiaceae, Prevotellaceae,Porphyromonadaceae, Pseudomonadaceae, Rikenellaceae, Rhizobiaceae and/orSutterellaceae.

The bacteria may optionally be of a genus selected from, e.g.,Abiotrophia, Achromobacter, Acidovorax, Acinetobacter, Actinobacillus,Actinomadura, Actinomyces, Aerococcus, Aeromonas, Anaerococcus,Anaplasma, Bacillus, Bacteroides, Bartonella, Bifidobacterium,Bordetella, Borrelia, Brevundimonas, Brucella, BurkholderiaCampylobacter, Capnocytophaga, Chlamydia, Citrobacter, Chlamydophila,Chryseobacterium, Clostridium, Comamonas, Corynebacterium, Coxiella,Cupriavidus, Delftia, Dermabacter, Ehrlichia, Eikenella, Enterobacter,Enterococcus, Escherichia, Erysipelothrix, Facklamia, Finegoldia,Francisella, Fusobacterium, Gemella, Gordonia, Haemophilus,Helicobacter, Klebsiella, Lactobacillus, Legionella, Leptospira,Listeria, Micrococcus, Moraxella, Morganella, Mycobacterium, Mycoplasma,Neisseria, Nocardia, Orientia, Pandoraea, Pasteurella, Peptoniphilus,Peptostreptococcus, Plesiomonas, Porphyromonas, Pseudomonas, Prevotella,Proteus, Propionibacterium, Rhodococcus, Ralstonia, Raoultella,Rickettsia, Rothia, Salmonella, Serratia, Shigella, Staphylococcus,Stenotrophomonas, Streptococcus, Tannerella, Treponema, Ureaplasma,Vibrio and/or Yersinia.

The bacteria may optionally be of a species selected from, e.g.,Abiotrophia defective, Achromobacter xylosoxidans, Acidovorax avenae,Acidovorax citrulli, Akkermansia muciniphila, Bacillus anthracis, B.cereus, B. subtilis, B. licheniformis, Bacteroides fragilis, Bartonellahenselae, Bartonella quintana, Bordetella pertussis, Borreliaburgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis,Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis,Burkholderia cepacia, Burkholderia genomovars, Campylobacter jejuni,Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci,Citrobacter koseri, Clostridium botulinum, Clostridium difficile, C.perfringens, C. tetani, Corynebacterium diphtherias, C. striatum, C.minutissimum, C. imitans, C. amycolatum, Delftia acidovorans,Enterobacter aerogenes, E. cloacae Enterococcus faecalis, Enterococcusfaecium, Escherichia coli, Francisella tularensis, Fusobacteriumnucleatum, Haemophilus influenzae, Helicobacter pylori, Klebsiellaoxytoca, K. pneumonia, Legionella pneumophila, Leptospira interrogans,Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeriaivanovii, Listeria monocytogenes, Micrococcus luteus, Morganellamorganii, Moraxella catarrhalis, Mycobacterium avium, M fortuitum, Mleprae, M peregrium, M tuberculosis, M ulcerans, Mycoplasma pneumoniae,Neisseria gonorrhoeae, N. lactamica, N. meningitidis, Nocardiaasteroids, Proteus mirabilis, Pseudomonas aeruginosa, Rhodococcus equi,Rhodococcus pyridinivorans,Rickettsia rickettsii, Salmonella typhi,Salmonella typhimurium, Serratia marcescens, Shigella sonnei,Staphylococcus aureus, S. capitis, S. epidermidis, S. haemolyticus, S.hominis, S. saprophyticus, Stenotrophomonas maltophilia, Streptococcusagalactiae, S. pyogenes, S. pneumonia, Treponema pallidum, Ureaplasmaurealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocoliticaand Yersinia pseudotuberculosis.

The virus may optionally be a DNA virus, and RNA virus or a retrovirus.It may optionally be a single stranded (ss) or a double stranded (ds)virus. More particularly, it may optionally be a ssDNA, dsDNA, dsRNA,ssRNA(positive strand), ssRNA (negative strand), ssRNA (reversetranscribed) or dsDNA (reverse transcribed) virus.

It may optionally be selected from one or more of the Herpesviridae,optionally selected from Simplexvirus, Varicellovirus, Cytomegalovirus,Roseolovirus, Lymphocryptovirus, and/or Rhadinovirus; the Adenoviridae,optionally selected from Adenovirus and/or Mastadenovirus;Papillomaviridae, optionally selected from Alphapapillomavirus,Betapapillomavirus, Gammapapilloma-virus, Mupapillomavirus, and/orNupapillomavirus; Polyomaviridae, optionally selected from Polyomavirus;Poxviridae, optionally selected from Molluscipoxvirus, Orthopoxvirusand/or Parapoxvirus; Anelloviridae, optionally selected fromAlphatorquevirus, Betatorquevirus, and/or Gammatorquevirus;Mycodnaviridae, optionally selected from Gemycircular-viruses;Parvoviridae, optionally selected from Erythrovirus, Dependovirus,and/or Bocavirus; Reoviridae, optionally selected from Coltivirus,Rotavirus, and/or Seadornavirus; Coronaviridae, optionally selected fromAlphacoronavirus, Betacoronavirus, and/or Torovirus; Astroviridae,optionally selected from Mamastrovirus; Caliciviridae, optionallyselected from Norovirus, and/or Sapovirus; Flaviviridae, optionallyselected from Flavivirus, Hepacivirus, and/or Pegivirus; Picornaviridae,optionally selected from Cardiovirus, Cosavirus, Enterovirus,Hepatovirus, Kobuvirus, Parechovirus, Rosavirus, and/or Salivirus;Togaviridae, optionally selected from Alphavirus and/or Rubivirus;Rhabdoviridae, optionally selected from Lyssavirus, and/orVesiculovirus; Filoviridae optionally selected from Ebolavirus, and/orMarburgvirus; Paramyxoviridae, optionally selected from Henipavirus,Heffalumpvirus, Morbilivirus, Respirovirus, Rubulavirus,Metapneumovirus, and/or Pneumovirus; Arenaviridae, optionally selectedfrom Arenavirus; Bunyaviridae, optionally selected from Hantavirus,Nairovirus, Orthobunyavirus, and/or Phlebovirus; Orthomyxoviridae,optionally selected from Influenzavirus A, Influenzavirus B,Influenzavirus C and/or Thogotovirus; Retroviridae, optionally selectedfrom Gammaretrovirus, Deltaretrovirus, Lentivirus, Spumavirus;Epadnaviridae, optionally selected from Orthohepadnavirus; Hepevirus;and/or Deltavirus.

The microbes may optionally be pathogenic or non-pathogenic. Apathogenic microbe, which may also be called a “pathogen”, may bedefined as a microbe that is able to cause disease in a host, such as aplant or animal. A pathogen may optionally be an obligate pathogen or anopportunistic pathogen.

The ability of a microbe to cause disease depends both on its intrinsicvirulence factors and on the ability of the host to fight off themicrobe. The distinction between non-pathogens and opportunisticpathogens is therefore not clear-cut, because, for example,immuno-compromised hosts will be susceptible to infection by microbesthat may be unable to infect a host with a healthy immune system.

For example, Neisseria gonorrhoeae is an obligate pathogen, Pseudomonasaeruginosa and Candida albicans are typically referred to asopportunistic pathogens, and Lactobacillus acidophilus andBifidobacterium bifidum are typically considered to be non-pathogens,and may be referred to as “commensal”.

Drugs, such as, an antimicrobial and/or an anti-inflammatory drug, mayalso create an environment in which a microbe will flourish as anopportunistic pathogen. Thus, the use of drugs may alter a microbiome.The method may therefore optionally involve analysing the microbiome,e.g., the mucosal microbiome, to analyse the response to a drug.

Pathogenic microbes may optionally be characterised by the expression ofone or more virulence factors, i.e. factors that allow or facilitateinfection of a host. Virulence factors may optionally be selected fromfactors that mediate cell adherence, cell growth, the ability to bypassor overcome host defence mechanisms, and/or the production of toxins.Toxins may be selected from exotoxins and endotoxins. The method mayoptionally involve analysing one or more virulence factors.

Commensal microbes are those which are part of the natural flora of ahuman or animal and which, in a balanced state, do not cause disease.

The community of microbes in a particular environment may be referred toas a “microbiome”. A microbiome may be a complex mixture of a vastnumber and vast variety of different microbes. The gastrointestinal (GI)microbiome is estimated to comprise over 100 trillion microbes thatrepresent at least several hundreds or even over a thousand differentspecies. The healthy human gut microbiota is dominated by theBacteroidetes and the Firmicutes, whereas, for example, Proteobacteria,Verrucomicrobia, Actinobacteria, Fusobacteria, and Cyanobacteria aretypically present in minor proportions.

The microbiome may vary from one environment to another within the samehuman or animal, so a person's gastrointestinal (GI) microbiome may bedifferent from that person's nasal microbiome. The GI microbiome mayfurther be divided into the different GI regions, such as, stomach,duodenum, jejunum, ileum, and/or colon. The lumen microbiome may alsodiffer from the mucosal microbiome. Each microbiome may also vary fromone individual to another. The disturbance of the normal microbiome maybe referred to as “dysbiosis”. Dysbiosis may cause, or be associatedwith, a disease, such as, any of the diseases mentioned herein. Themethod may optionally involve the analysis of a microbiome to analysedysbiosis. The GI microbiome may also be referred to as the “gut flora”.

The microbiome may change during pregnancy, so an analysis of the female(human or animal) microbiome may allow an analysis of pregnancy.Dysbiosis in pregnancy is associated with complications, such as, anincreased risk of premature birth.

Dysbiosis may involve the presence of one or more types of microbes thatare normally, or were previously, absent from a particular microbiome.However, more commonly, dysbiosis may involve a relative increase in theproportion of one or more particular microbes, and/or a relativedecrease in the proportion of one or more particular microbes.

As mentioned above, the mucosa comprises layers of mucus. Microbes, suchas bacteria, may adhere to and/or partially or fully infiltrate themucus layer. The microbial adherence and/or proliferation may beinfluenced by carbohydrate modifications present on mucins; byantimicrobial agents, such as, host-derived antimicrobial peptides; bydrugs; by diet; and/or by toxins, such as, toxins produced by(pathogenic) microbes.

The mucosal (epithelial) surface beneath the mucus layer is free ofmicrobes in at least about 80% of healthy humans. The thickness of themucus layer and its spread may vary, for example, they may decrease withincreasing severity of inflammation. Under certain conditions, forexample, in a disease, microbes may infiltrate and/or adhere to themucus layer, the epithelium and/or the LP. For example, bacteria maytypically be found within the mucus of biopsy specimens from subjectswith ulcerative colitis, SLC, and/or acute appendicitis. Theconcentration of microbes within the mucus layer may inversely correlateto the numbers of leucocytes.

The term “mucosal microbiome” is used herein to denote the microbiomewhich is associated with the mucosa, including the microbiome that hasinfiltrated the mucosa and the microbiome that is associated with (forexample, through adhesion or partial or full infiltration) with themucus layer.

The method may optionally involve the analysis of an infection, e.g.,the diagnosis of an infection, analysis of the genotype or phenotype ofthe infection-causing microbe, monitoring of progression of infection,and/or monitoring of treatment response to infection.

The method may optionally involve the analysis of vaccination. This may,e.g., involve analysing a target prior to and after vaccination.Optionally, the subject may be challenged after vaccination with themicrobe against which the vaccination is aimed, and a suitable targetmay then be analysed to determine whether, or at what level, the microbeis present. The presence or level of the microbe may be indicative ofthe success of vaccination, e.g., the absence or presence at low levelsof the microbe may be indicative of successful vaccination, whereas thepresence, or presence at high levels of the microbe may be indicative ofthe vaccine being deficient or ineffective.

The mucosal membrane may be considered to be a protective layerresponsible for trapping pathogens in the human body.

The mucosa lines several passages and cavities of the body, particularlythose with openings exposed to the external environment, including theoral-pharyngeal cavity, gastrointestinal (GI) tract, respiratory tract,urogenital tract, and exocrine glands. Thus, the mucosa may optionallybe selected from Bronchial mucosa, Endometrium (mucosa of the uterus),Esophageal mucosa, Gastric mucosa, Intestinal mucosa (gut mucosa), Nasalmucosa, Olfactory mucosa, Oral mucosa, Penile mucosa and/or Vaginalmucosa.

Broadly speaking, the mucosa comprises a mucus layer (the inner mucuslayer); an epithelium; a basement membrane, a Lamina propria (LP), whichis a layer of connective tissue; and a Muscularis mucosae, which is athin layer of smooth muscle. Thus, the term “mucosa” is used herein torefer to this entire complex, unless stated otherwise and the term“mucosal membrane” is used interchangeably with the term “mucosa”. Themucosa may also be covered by a further, outer mucus layer, which istypically more loosely associated therewith. Any reference herein to a“mucosa” may include reference to this further, outer mucus layer.Adjacent to the mucosa is the submucosa.

The inner mucus layer may be degraded by microbes. For example, mucinmonosaccharides may be used by bacteria, e.g., commensal bacteria, as anenergy source. Therefore, continuous renewal of the inner mucus layer isvery important.

The epithelium is a single or multiple layer(s) of epithelial cells. Theepithelium may comprise, for example, intra-epithelial lymphocytes(IELs), endocrine cells, goblet cells, enterocytes and/or Paneth cells.

The basement membrane may comprise various proteins, particularlystructural or adhesive proteins, such as, laminins, collagens, e.g.,collagen IV, proteoglycans, and/or calcium binding proteins such asfibulin.

The Lamina propria is connective tissue which may comprise, for example,plasma cells, eosinophils, histiocytes, mast cells and/or lymphocytes.Neutrophils are generally absent in the Lamina propria of healthyhumans.

As discussed below, the mucosa may also comprise, for example, antigenpresenting cells (APCs) and microfold cells (M-cells). The mucosa mayinclude one or more distinct types of regulatory immune cells, includingintestinal intraepithelial lymphocytes (IELs), Foxp3(+) regulatory Tcells, regulatory B cells, alternatively activated macrophages,dendritic cells, and/or innate lymphoid cells.

The mucosa typically secretes mucus, which forms a mucus layer betweenthe mucosal epithelium and the lumen. The mucus layer may have aprotective function. A major constituent of mucus are mucins, which areproduced by specialized mucosal cells called goblet cells. Mucins areglycoproteins characterized mainly by a high level of O-linkedoligosaccharides. The level to which the protein moiety is linked to thecarbohydrate moieties, as well as the precise identity of thecharbohydrate moieties, may vary significantly.

Mucosa establish a barrier between sometimes hostile externalenvironments and the internal milieu. However, mucosae are alsoresponsible for nutrient absorption and waste secretion, which require aselectively permeable barrier. These functions place the mucosalepithelium at the centre of interactions between the mucosal immunesystem and luminal contents, including dietary antigens and microbialproducts. Thus, many physiological and immunological stimuli triggerresponses in the mucosa. Dysfunctional responses may contribute todisease.

The mucosal immune system is a localized and specific immuneorganisation. The mucosal immune system at different organs sharesimilar anatomical organization and features. The GI mucosal immunesystem is best understood, and is discussed below for illustrativepurposes. The GI mucosal immune system is composed of three majorcompartments: the epithelial layer; the lamina propria (LP); and themucosal-associated lymphoid tissue (MALT), which, in the GI tract, maybe referred to as gut-associated lymphoid tissue, and which comprisesPeyer's patches and isolated lymphoid follicles.

Dendritic cells may project dendrites into the epithelium to uptakeantigens and migrate to the LP, secondary lymphoid tissue and draininglymph nodes, where they prime naive T cells. Microfold cells (M-cells),located in the epithelium of Peyer's patches, may pass the antigens todendritic cells, macrophages and other antigen presenting cells. Naive Tcells in secondary lymphoid tissues may become activated after beingprimed by antigen presenting cell and home to LP (called LPLs) orinfiltrate into inflamed epithelium.

The gastrointestinal (GI) tract can be divided into four concentriclayers that surround the lumen in the following order: (i) Mucosa; (ii)Submucosa; (iii) Muscular layer; and (iv) Adventitia or serosa.

Thus, the GI mucosa is the innermost layer of the gastrointestinaltract. This layer comes in direct contact with digested food. In the GImucosa, the epithelium is responsible for most digestive, absorptive andsecretory processes, whereas the Muscularis mucosae aids the passing ofmaterial and enhances the interaction between the epithelial layer andthe contents of the lumen by agitation and peristalsis.

GI mucosae are highly specialized in each organ of the GI tract to dealwith the different conditions. The most variation may occur in theepithelium.

Different types of mucosa differ from one another and the inventors haveshown that the method of the various embodiments described herein mayoptionally be used, e.g., to distinguish between different types ofmucosa, e.g. vaginal, nasal and oral.

FIG. 2 illustrates various different mucosa or mucosal membranes whichare present in the human body.

Mucosal membranes 200 comprise a layer of epithelial tissue which linesall passages in the human body that are open to the externalenvironments including the nose and parts of the digestive, urogenitaland respiratory tracts. Mucosal membranes typically act as a protectivebarrier to trap pathogens such as bacteria, viruses and fungi. Forexample, the mucosae of oral, respiratory and urogenital tracts arecomposed of epithelial tissue and underlying lamina that are directlyexposed to the external environment, making them a primary site ofinnate and acquired protection against host infection.

As shown in FIG. 2, mucosal membranes are present in the mouth, pharynx,and respiratory system 201, as well as in the gastro-intestinal tract202 and the urogenital tract 203, and include the endometrium,intestinal, gastric, oral, vaginal, esophageal, gingival, nasal, buccaland bronchial membranes.

Studies as part of the human microbiome project have revealed thatcolonization by different microbial species within the mucosa has animmense impact upon human health and disease. As discussed elsewhereherein, many diseases (e.g. cancer, infections, etc.) are associatedwith the mucosa. Host-microbiota interactions at mucosal surfaces havean important impact not only on pathology and disease, but also onhealth states. For example, commensal vaginal microbiota excreteantimicrobial compounds and metabolites into the cervicovaginal mucosathat modulates both its physical and immunological properties. Duringpregnancy, this mechanism is thought to provide protection againstpathobiont colonisation of the reproductive tract, which is a majorcause of preterm birth. Nasal mucosal surfaces are key modulators ofallergic inflammation and airway obstruction pathologies such as asthma.

As such, the mucosal membrane is an easily accessible and highlyclinically relevant sample to analyse, e.g., to diagnose diseases, e.g.,microbial and/or cancerous associated diseases, etc., and mucosaldiagnostics represents an important field that has wide clinicalapplications.

As shown in FIG. 3, a typical mucosal membrane may be present in a lumen300 and may include mucus 301, bacteria 302, lymphatic vessels 303,blood vessels 304, mucosal glands 305, and submucosa 306. As illustratedby FIG. 3, the biological tissue of the mucosa itself, e.g. mucus 301,and/or bacteria 302 present in or associated with the mucosa representpotential analytes/biomarkers. For example, membrane lipids, and/orinflammatory markers of the mucosa, and/or complex lipids and/orsignalling molecules of intact bacteria cells represent potentialanalytes/biomarkers.

The method according to various embodiments may involve the analysis ofa mucosal target, e.g., on a swab or biopsy. Optionally, the method mayinvolve the analysis of a mucosal target to analyse the cellularcomposition of the mucosa; to analyse a disease; to analyse the responseto a drug; to analyse the response to a particular food, diet, and/or achange in diet; to analyse a mucosal microbe; to analyse a microbialinteraction with the mucosa; and/or to analyse the mucosal microbiome.

The analysis of the cellular composition of a mucosa, may, e.g., analysethe presence or absence and/or proportion of one or more cell types,which may optionally be selected from any of the cell types listedherein. Optionally, the method may involve the analysis of MALT and/or aPeyer's patch. Optionally, the method may involve the analysis of thephenotype and/or genotype of one or more cell types, which mayoptionally be selected from any of the cell types listed herein.

Optionally, the method may involve the analysis of a change in themucosa, which may optionally be a change in, e.g., the cellularcomposition of the mucosa, the microbial interaction(s) with the mucosa,and/or the mucosal microbiome. By a “change” in the mucosa is meant thatthe mucosa is different from how it would typically present in a healthysubject; that it is different in one location compared to anotherlocation within the same subject; and/or that it is different from howit was when it was analysed at an earlier point in time. A change in themucosa may optionally, for example, be caused by, or associated with, adisease, the response to a substance, such as a drug, and/or theresponse to a food, diet, and/or diet change.

A disease may optionally be selected from an autoimmune disorder, aninflammatory disease, tropical sprue, a food intolerance, an infection,a cancer, and/or any of the of the disorders mentioned herein.

More particularly, the disease may optionally be selected from, forexample, asthma, Coeliac disease, gastritis, peptic duodenitis,Gluten-sensitive enteropathy; allergy and/or intolerance to an allergen,e.g. to milk, soy, tree nut(s), egg, wheat, meat, fish, shellfish,peanut, seed, such as sesame, sunflower, and/or poppy seeds, garlic,mustard, coriander, and/or onion; Hashimoto's thyroiditis; Irritablebowel syndrome; Graves's disease; reactive arthritis; psoriasis;multiple sclerosis; Systemic lupus erythematosus (SLE or lupus);ankylosing spondylitis; progressive systemic sclerosis (PSS);glomerulonephritis; autoimmune enteropathy; IgA deficiency; commonvariable immunodeficiency; Crohn's disease; colitis, such as,lymphocytic colitis, collagenous colitis and/or ulcerative colitis;diffuse lymphocytic gastroenteritis; ulcer; intestinal T-cell lymphoma;infection, e.g., pharyngitis, bronchitis, and/or infection with amicrobe selected, for example, from Giardia, Cryptosporidium,Helicobacter and/or any of the other microbes mentioned herein; and/orcancer, details of which are discussed elsewhere herein.

The method may, e.g., optionally involve the analysis of the interactionof the mucosa with microbes, or a change in the mucosa caused by, orassociated with, such an interaction. Optionally, the interaction may,e.g., be the translocation of microbes into the mucosa, e.g., thetranslocation of commensal bacteria. The method may, e.g., optionallyinvolve the analysis of the mucosal microbiome, or a change in themucosa caused by, or associated with, the mucosal microbiome. The methodmay, e.g., optionally involve the analysis of an infection, or a changein the mucosa caused by, or associated with, an infection. The analysisof microbes, a microbial interaction, infections and/or the microbiomeare also discussed elsewhere herein.

As mentioned above, IELs are a normal constituent of the smallintestinal mucosa. They play a significant role in immune surveillanceand activation. In healthy humans, the vast majority of IELs are ofT-cell type and express an α/β-cell receptor on their surface. It isgenerally accepted that healthy humans have no more than about 20lymphocytes per 100 epithelial cells in the intestinal mucosa.

An increased number of lymphocytes in a mucosal specimen may optionallybe indicative of a change, such as, a disease, the response to a drug,and/or a microbial change. The term “elevated” or “increased” levels ofIELs is therefore used to refer to more than 20 IELs per 100 epithelialcells in the intestinal mucosa, optionally at least 22, 24, 25, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 65, 70,75 or 80 IELs per 100 epithelial cells in the intestinal mucosa.

The gamma-delta receptor of T lymphocytes is not expressed by more than2-3% of T lymphocytes in normal conditions. An increase in thepercentage of T lymphocytes expressing this receptor may therefore beindicative of a change, such as, a disease, the response to a drug,and/or a microbial change. The method may therefore involve determiningthe presence or percentage of T lymphocyte gamma-delta receptorexpression. For example, in coeliac disease 20-30% of mucosal Tlymphocytes may express this receptor.

Thus, the method may optionally involve the analysis of lymphocytes in atarget, which may optionally be T lymphocytes, e.g. gamma-deltareceptor-positive T lymphocytes. Optionally, a target may be analysedfor an increase or decrease in the number of lymphocytes. Optionally,the phenotype and/or genotype of the lymphocytes may be analysed.

Polymorphonuclear leukocytes (PMN), also called neutrophils, are themost abundant leukocyte population in the blood, comprising 50-60% ofthe circulating leukocytes (25×109 cells). PMN are critical componentsof the innate immune response that are essential in protecting the host,e.g., from microbial pathogens, while also minimizing deleteriouseffects mediated by dying or injured cells.

PMN may perform a variety of antimicrobial functions such asdegranulation and phagocytosis. They are uniquely capable of forminglarge amounts of reactive oxygen species and other toxic molecules thatmay weaken and/or destroy pathogens. Upon PMN contact with invadingmicrobes, reactive oxygen species may be generated in an oxidative burstby an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase

PMN may also possess different pools of intracellular granules thatcontain antimicrobial peptides, such as, a-defensins and/orcathelicidins; myeloperoxidase; hydrolytic enzymes, such as, lysozyme,sialidase, and/or collagenase; proteases, such as, cathepsin G;azurocidin, and/or elastase; cationic phospholipase; and/or metalchelators such as lactoferrin. Such granules may be released uponcontact with microbes.

PMN may also be capable of imprinting the tissue with neutrophilextracellular traps (NETs). NETs may be composed of nuclear contents(DNA and chromatin) mixed with toxic molecules from intracellulargranules and the cytosol. Invading microorganisms may be sequestered inthese NETs and effectively destroyed.

During intestinal inflammation, resident monocytes contribute to therecruitment of neutrophils through production of macrophage-derivedchemokines. Neutrophils present in the blood sense the chemoattractantgradient and traverse the vascular endothelium to reach the intestinallamina propria. In this manner, neutrophils are recruited to sites ofinfection or inflammatory stimuli within minutes. The response typicallypeaks by 24-48 hours. Under certain physiological or pathologicalconditions, neutrophils may cross the epithelium into the intestinallumen.

At inflammatory sites, neutrophils may selectively release monocytechemoattractants, such as CAP18, cathepsin G, and/or azurocidin. Thus,shortly after arrival of PMN to the mucosa, macrophages are recruitedfor a second-wave inflammatory response that ensues for the next severaldays.

Thus, the method may optionally involve the analysis of neutrophils in atarget. Optionally, the presence of reactive oxygen species and/orneutrophils generating reactive oxygen species in a target may beanalysed. Optionally, the presence of NETs and/or neutrophils generatingNETs in a target may be analysed. Optionally, the presence of monocytechemoattractants and/or neutrophils generating monocyte chemoattractantsin a target may be analysed.

According to various embodiments, microbial, e.g., bacterial, and/oranimal, e.g., human mucosal membrane analytes may be characterised, e.g.using ambient mass spectrometry based techniques such as the desorptionelectrospray ionisation (“DESI”) technique and the rapid evaporativeionisation mass spectrometry (“REIMS”) technique.

As illustrated by FIG. 4, these analytes (e.g., membrane lipids andinflammatory markers of the mucosa, and complex lipids and signallingmolecules of intact bacteria cells) can be useful in identifying anumber of clinical disorders.

Accordingly, various embodiments are directed to the development of areal time point of care (“POC”) diagnostic method to investigate variousclinical disorders. In particular, various embodiments are directed tomass spectrometry (“MS”) based real-time point of care (“POC”)techniques.

For example, infections such as pharyngitis, bronchitis, and/orinfections with any of the microbes mentioned herein etc. can beidentified e.g. by analysing, e.g., identifying microbes.

Changes in the microbiome can also be analysed, e.g., detected, e.g., byidentifying microbes, and by way of example, determining a change in themicrobiome of a pregnant patient can be used to identify those patientswho are at an increased risk of having a pre-term or premature deliveryduring pregnancy.

Furthermore, the various analytes taken from mucosal membranes, e.g.biomarker profiling, can be used to identify various immunologicaldisorders (e.g., asthma, allergies) as well as to identify cancer andpre-cancerous states.

As further illustrated by FIG. 5, metabolomic profiling of analytes fromvarious mucosal membranes using swabs can be useful in identifying anumber of clinical disorders. For example, allergies may be identified,e.g., by identifying inflammatory mediators (eicosanoids) such asprostaglandins (PGD2), leukotriends, histamine, etc. Inflammation (suchas pharyngitis, angina, etc.) may be identified, e.g., by identifyingmicrobial, e.g., bacterial secondary metabolites, lipids, etc. frombacteria such as streptococcus sp., staphylococcus sp., haemophilus sp.,etc. Pre-term delivery may also be identified, e.g. by identifyinghealthy (e.g. comprising a stable lactobacilli environment includinge.g., L. crispatus dominant, L. iners dominant, and/or L. gasseri mix,etc.) or unhealthy mucosa (e.g. comprising an overgrowth of pathogensincluding, e.g., Escherchia coli, Atopobium vaginae, Peptostreptococcus,and/or Bacteroides sp., etc.).

According to various embodiments, mucosal diagnostics enablenon-invasive direct sampling of the mucosa from patients at a clinicalpoint of care.

According to various embodiments, analytes may be obtained from mucosalmembranes using, e.g., a “standard medical swab”, i.e., a standardcollection or sampling device for mucosa that can be used in clinicalmicrobiology (e.g. to take microbiological cultures), gene and drugtesting, etc. The “standard medical swab” may comprise, for example, apad of cotton, i.e. the standard sample collecting device for mucosa. Aswill be discussed in more detail below, the medical swab may be wettedor otherwise functionalised.

For clinical analysis, the swabs may be wiped over or into an infectedarea, e.g. to sample microbe rich body fluid, such as, sanies, and/orthe mucosa itself. For conventional microbial analysis, the swab maythen be placed into a sterile tube which may contain a buffer solutionor transport media for storage before the tube is sent to a laboratoryfor analysis such as microscopy and/or culture-based characterisation ofthe microbial content. For example, a laboratory receiving the tube maywipe the smear content across a culture medium such as an agar plate.The culture medium may then be incubated to allow organisms present togrow. Microbial identification may then be performed under a microscope.Any organisms present in the sample may also be identified, e.g., bysequence analysis, e.g.,16S gene-sequencing of bacteria, and/or by usingmatrix-assisted laser desorption ionisation (“MALDI”) mass spectrometryand then comparing the mass spectra with a commercially availabledatabase.

FIG. 6 illustrates a microbe identification workflow and shows sampling601 an analyte using a swab and then transporting 602 the swab to aspecialist laboratory for microbe culturing 603 and further analysis. Asshown in FIG. 6, such culture based analysis may comprise imaging usinga microscope 604 and/or Matrix Assisted Laser Desorption Ionisation(“MALDI”) Mass Spectrometry (“MS”) 605 followed by statistical analysis606, etc. 16s rRNA sequencing 607 is a culture independent analysismethod.

Although easy to handle, the current analysis of medical swabs fordiagnostic purposes is culture-dependent and involves a relatively timeconsuming and relatively costly workflow. Diagnosis ofpathogen-associated diseases and appropriate treatment is thereforeassociated with considerable delay. Furthermore, around 95% of bacteriacannot be cultured for analysis. As such, conventional methods arelimited by their qualitative nature, time required to obtain results andinability to assess host response to the presence or absence of specificmicrobiota.

Sequencing of bacterial 16S rRNA gene or analysis of samples bymatrix-assisted laser desorption ionization (MALDI) mass spectrometry(MS) are also time consuming and costly.

Various embodiments which are described in more detail below provide afast and direct way to investigate clinical samples from mucosalmembranes, e.g. by identifying microbes and/or biomarkers characteristicof specific clinical disorders in mucosal samples, thereby permittingfaster diagnoses and treatment of patients.

Various embodiments are directed to real time rapid and direct analysisof analytes present on a swab using ambient mass spectrometry. Ambientionisation mass spectrometry based techniques may be employed for directanalysis of the sample surface. A sample may be analysed in its nativestate with minimal or no prior sample preparation.

Various embodiments permit rapid acquisition of detailed mass spectralmetabolic fingerprints from a wide variety of biological materials,including intact bacteria, without the need of extraction or extensivesample preparation protocols.

In particular, Desorption Electrospray Ionisation (“DESI”) has beenfound to be a particularly useful and convenient method for the realtime rapid and direct analysis of analytes present on a swab. Desorptionelectrospray ionisation (“DESI”) allows direct and fast analysis ofsurfaces without the need for prior sample preparation. The techniquewill now be described in more detail with reference to FIG. 7.

As shown in FIG. 7, the desorption electrospray ionisation (“DESI”)technique is an ambient ionisation method that involves directing aspray of (primary) electrically charged droplets 701 onto a surface 702with analyte 703 present on the surface 702 and/or directly onto asurface of a sample 704. The electrospray mist is pneumatically directedat the sample by a sprayer 700 where subsequent splashed (secondary)droplets 705 carry desorbed ionised analytes (e.g. desorbed lipid ions).The sprayer 700 may be supplied with a solvent 706, gas 707 such asnitrogen, and voltage from a high voltage (“HV”) source 708. Afterionisation, the ions travel through air into an atmospheric pressureinterface 709 of a mass and/or ion mobility spectrometer or massanalyser (not shown), e.g. via a transfer capillary 710.

The desorption electrospray ionisation (“DESI”) technique allows forambient ionisation of a trace sample at atmospheric pressure with littlesample preparation. The desorption electrospray ionisation (“DESI”)technique allows, for example, direct analysis of biological compoundssuch as lipids, metabolites and peptides in their native state withoutrequiring any advance sample preparation.

Embodiments described herein relate to directly analysing medical swabsusing desorption electrospray ionisation (“DESI”) mass spectrometry.According to various embodiments chemical signature identification ofspecific microbes, e.g., bacteria and/or biomarkers on the surface ofthe swabs is possible within a relatively short period of time.

Various specific embodiments relate to the rapid diagnosis of infectionsand/or dysbiosis, e.g., associated with preterm (premature) delivery(and these results may optionally be compared with standard microbialtesting).

Further embodiments relate to a real-time rapid medical swab analysisusing desorption electrospray ionisation (“DESI”) mass spectrometry toreveal pathogenic and/or inflammatory metabolomic markers.

Furthermore, various chemically modified swabs for use with desorptionelectrospray ionisation (“DESI”) mass spectrometry are disclosed. Thesehave been found to exhibit an improved sensitivity compared withconventional (non-modified) swabs.

It has also been found that a significant enhancement in signalintensity can be obtained by rotating or continuously rotating a swabwhilst analysing the swab using the desorption electrospray ionisation(“DESI”) technique.

Desorption electrospray ionisation (“DESI”) mass spectrometry analysisof medical swabs is relatively simple, e.g. compared to liquidextraction based mass spectrometry techniques including high performanceliquid chromatography (“HPLC”) mass spectrometry, as no samplepreparation steps are required prior to analysis and ionisation occursdirectly from the (rotatable) medical swab. Desorption electrosprayionisation (“DESI”) mass spectrometry analysis of medical swabs is alsoan improvement over to so-called “touch spray” (“TS”) mass spectrometry,which is limited in terms of the reproducibility of and control over thespray formation and spray stability due to strong evaporation and dryingof swabs.

FIGS. 8A-C illustrate a desorption electrospray ionisation (“DESI”) massspectrometry setup for swab analysis according to various embodiments.As shown in FIG. 8A, a desorption electrospray ionisation (“DESI”)sprayer 800 and a mass spectrometry inlet capillary 801 may bepositioned adjacent to a medical swab 802. The sprayer 800 may beprovided with a gas supply 803, a power/solvent supply 804, and may beprovided on a movable sprayer stage 805. The swab 802 may be provided ona movable swab stage 806.

As shown in FIG. 8B, the swab 802 may be rotated in order to accessdifferent portions of the analyte on the swab 802. The arrow in FIG. 8Bshows the direction of motion of the swab according to an embodiment.

Initial experiments optimised the swab-inlet geometry, tip-sample anglesand distances, and rotation speeds and provided high repeatability forthe desorption electrospray ionisation (“DESI”) mass spectrometryanalysis. It was found that the optimum parameters for this setupinclude a swab-capillary distance of around 1-2 mm, a sprayer-swabdistance of around 1-2 mm, a sprayer voltage of around 4.3 kV, a solventflow rate of around 10 μL/min and a nebuliser gas pressure of around 7bar.

FIG. 8C illustrates a number of swab positions and geometries inaccordance with various embodiments.

Various embodiments relate to the application of desorption electrosprayionisation (“DESI”) mass spectrometry to direct analysis of standardmedical swabs, thereby permitting rapid assessment of perturbations ofmucosal surface chemistry. As such, various embodiments relate to thedevelopment of a non-invasive point of care diagnostic technique, e.g.directed toward detection of diseases such as the detection ofinfections, dysbiosis, cancer and/or inflammatory diseases, and/or anyof the other diseases mentioned elsewhere herein.

Medical swabs were analysed by desorption electrospray ionisation(“DESI”) mass spectrometry with the intention of extracting chemicalinformation relevant to patient care in a non-invasive procedure. Inthis context, desorption electrospray ionisation (“DESI”) massspectrometry represents a fast and direct method for metabolomicprofiling of different mucosal membrane models or membranes (e.g. nasal,vaginal, oral) by desorbing and analysing molecules from the surface ofstandard medical cotton swabs.

Since the design of the swab for each clinical application may vary,with appropriate shape and materials being chosen for each type ofapplication, different shapes of commercial available swabs were tested.

A study was performed in which vaginal mucosa (n=25 pregnant, n=25non-pregnant), nasal mucosa (n=20) and oral mucosa (n=15) were sampledwith medical rayon swabs from patients. Medical cotton swabs sold asTranswab® Amies (MWE medical wire, Wiltshire, UK) were used for samplingmucosal membranes which were then transferred to a sterile tube withoutbuffer or storage medium solution and were stored at −80° C. in afreezer.

FIG. 9 highlights the sampling points of analysed mucosal membranescollected from the urogenital tract, oral and nasal cavity with amedical cotton swab 900. As illustrated by FIG. 9, the surface of themedical swab 900 was directly analysed by desorption electrosprayionisation (“DESI”) mass spectrometry without prior sample preparationprocedures.

Desorption electrospray ionisation (“DESI”) mass spectrometryexperiments were performed using a Xevo G2-S Q-TOF® mass spectrometer(Waters®, Manchester, UK). The desorption electrospray ionisation(“DESI”) source comprises an electronic spray emitter 901 connected witha gas 902, solvent 903 and power supply 904 and an automatic rotatableswab holder device 905 with adjustable rotation speed.

For the desorption electrospray ionisation (“DESI”) mass spectrometryanalysis the medical swab 900 was positioned orthogonally to and infront of an inlet capillary 906 connected to the mass and/or ionmobility spectrometer atmospheric pressure interface 907. A mixedmethanol:water solution (95:5) spray solvent was used at a flow rate ofaround 10 μL/min for desorption of the sample material. Nitrogen gas ataround 7 bar and a voltage of around 3.4 kV were also provided to thesprayer 900.

The mucosa was absorbed from the surface of the rotated swabs by gentlydesorbing molecules with charged droplets of the organic solvent, anddesorbed ions (e.g. lipids) were subsequently transferred to the massand/or ion mobility spectrometer.

Full scan mass spectra (m/z 150-1000) were recorded in negative ionmode. Mass spectral data were then imported into a statistical analysistoolbox and processed. For data analysis and extraction of specificmolecular ion patterns, an unsupervised principal component analysis(“PCA”) as well as a recursive maximum margin criterion (“RMMC”)approach were applied to improve supervised feature extraction and classinformation with leave one out cross validation (“CV”) to determineclassification accuracy within the data set.

FIGS. 10A and 10B show the results of desorption electrospray ionisation(“DESI”) mass spectrometry analysis of swabs, and multivariatestatistical analysis including principal component analysis (PCA) andrecursive maximum margin criterion (RMMC), which were used to identifylipid patterns characteristic of different mucosal models.

FIG. 10A shows averaged negative-ion mode desorption electrosprayionisation (“DESI”) mass spectra from vaginal, oral and nasal mucosarecorded using a Xevo G2-S Q-Tof ® mass spectrometer.

FIG. 10B shows a principal component analysis (“PCA”) and a maximummargin criterion (“MMC”) score plots for vaginal (n=68), oral (n=15) andnasal (n=20) mucosa acquired with desorption electrospray ionisation(“DESI”) mass spectrometry.

As shown in FIG. 10A, unique lipid patterns were observed betweendifferent mucosal membrane models. The spectra for vaginal mucosa andoral mucosa featured predominately glycerophospholipids, e.g.,[PS(34:1)-H]⁻ having a mass to charge ratio (“m/z”) of 760.4,[PS(36:2)-H]⁻ having a m/z of 788.5 and [PI (36:1)-H]⁻ having a m/z of863.4.

As shown in FIG. 10A, nasal mucosa featured mainly chlorinated adducts[PC(36:2)+Cl]⁻ m/z 820.5, [PC(34:1)+Cl]⁻ m/z 794.5 and [PI(36:2)-H]⁻ m/z826.4 in the m/z 700-900 range.

A characteristic feature of vaginal mucosa was deprotonated cholesterolsulphate at a m/z of 465.3, which was consistently observed to be themost dominant peak in the spectrum. Chemical assignment of this peak wasconfirmed by tandem mass spectrometry experiments. This compound is animportant component of cell membranes with regulatory functionsincluding a stabilizing role, e.g., protecting erythrocytes from osmoticlysis and regulating sperm capacitation.

Leave-one-patient-out cross validation of the multivariate modelcontaining spectra obtained by the analyses of three mucosal modelsresulted in a high classification accuracy. This show that MS basedprofiling of different mucosal membranes allows stratification ofpatients based upon bacterial diversity.

Similarly, FIG. 11 shows Fourier transform mass spectrometry (“FTMS”)mass spectral data obtained from vaginal, oral and nasal mucosa onmedical cotton swabs in negative ion mode in the mass range of m/z150-1000. Again, different metabolic signatures were observed in eachmucosal membrane model.

In total, 300 to 1000 spectral features found without isotopes andadducts including small human primary metabolites such as cholesterolsulphate, bacterial secondary metabolites including lactate as well asglycerophospholipids were tentatively identified by exact mass, isotopecluster distribution and tandem mass spectrometry experiments in themucosal membrane.

FIG. 12 shows a desorption electrospray ionisation (“DESI”) massspectrum relating to a pregnant vaginal mucosal membrane in more detailwhich was obtained in negative ion mode using a medical cotton swab. Theurogenital mucosa was found to produce cholesterol sulphate [M−H]⁻ at am/z of 465.41 as the most abundant lipid species as well as a differentglycerophosholipids species such as glycerophosphoethanolamine (PE)[PE(40:7)-H]⁻ at a m/z of 788.50, glycerophosphoserine (PS)[PS(34:1)-H]⁻ at a m/z of 760.50 and glycerophosphoinositol (PI)[PI(36:1)-H]⁻ at a m/z of 863.58. As shown in FIG. 12, chemicalassignment of the cholesterol sulphate peak was confirmed by tandem massspectrometry experiments.

The mass spectral data of FIG. 11 were further processed using mediannormalization, background subtraction, Savitzky-Golay peak detection,peak alignment and log-transformation. Following data processing,multivariate statistical analysis was applied on the data set tocharacterise distinct mucosa models based on their metabolic profile.Multivariate statistical analysis tools including principal componentanalysis (PCA) and maximum margin criterion (MMC) were used to analysethe data set.

As shown in FIG. 11, the PCA score plot as well as the MMC score plotreveal a separation of the different mucosal membrane types within thefirst two components with a prediction accuracy between 92-100% obtainedby leave one out cross validation.

It will be appreciated that analysis according to various embodimentsresults in characteristic profiles for the various sample types that canbe clearly distinguished e.g., by using PCA, MMC and/or leave one outcross validation analyses. These results show the use of desorptionelectrospray ionisation (“DESI”) mass spectrometry to characterise humanmucosal membrane models, e.g. based on their metabolic signaturesexcreted by characteristic bacteria, as a fast bacterial identificationmethod, e.g., compared to 16S rRNA sequencing.

Further embodiments are contemplated wherein chemical biomarkers inhuman mucosal membranes may be measured, which are reliable predictorse.g. in the cases of dysbiotic, inflammatory, cancerous and/orinfectious diseases.

Pregnancy involves major changes in circulating hormone (e.g. estrogenand progesterone) levels as well as their secondary metabolites.Moreover, pregnancy is associated with a reduction in vaginal microbialdiversity and an increase in stability. As described below, differencesin the chemical signature of vaginal mucosa in normal pregnancy and thenon-pregnant state can be readily determined using desorptionelectrospray ionisation (“DESI”) mass spectrometry according to variousembodiments.

A clinical set of pregnant (n=22, in a gestational age between 26 and 40weeks) and non-pregnant mucosal membrane (n=22) were evaluated in moredetail in order to reveal metabolic signature differences caused by achange in the vaginal microbiome during pregnancy. Desorptionelectrospray ionisation (“DESI”) mass spectrometry spectra were acquiredfrom both groups in negative ion mode in the mass range of m/z 150-1000.A number of different metabolites were detected in the vaginal mucosalmembrane.

FIG. 13A shows averaged desorption electrospray ionisation (“DESI”) massspectra from pregnant and non-pregnant group acquired in the negativeion mode in the mass range m/z 150-1000. A comparison of the averagedspectra shown in FIG. 13A shows spectral differences betweennon-pregnant and pregnant mucosa metabolic profiles, especially in thelipid mass range from m/z 550-900.

Further data analysis comprising unsupervised PCA and RMIVIC analysisrevealed clear separation between the two groups with a high (>80%)classification accuracy as determined using leave-one-out crossvalidation.

FIGS. 13B and 13C show the results of multivariate statistical analysisof pregnant (n=22) and non-pregnant (n=22) vaginal mucosal membraneusing desorption electrospray ionisation (“DESI”) mass spectrometry.

FIG. 13B shows principal component analysis and discriminatory analysisusing RMMC and FIG. 13C shows analysis with leave-one-outcross-validation.

FIG. 13D shows box plots which indicate significant differences in theabundance of selected lipid peaks between non-pregnant and pregnantvaginal mucosal membrane mainly in the range from m/z 550-1000 obtainedby Kruskal-Wallis ANOVA, p<0.005.

As shown in FIG. 13E, using RMIVIC both groups separate well in the RMMCspace with a high (>80%) classification accuracy according to distinctmetabolic signatures obtained by leave-one-patient-out cross validation.

Clinical studies have shown that vaginal microbial, e.g., bacterialdiversity is associated with specific vaginal mucosal metabolites. Forexample, during healthy pregnancy the vaginal mucosa is colonized mainlyby the Lactobacillus species. However, importantly, a shift towardsvaginal dysbiosis during pregnancy may be a causal trigger for pretermbirth.

Using the desorption electrospray ionisation (“DESI”) mass spectrometrybased technique disclosed herein allows females, e.g., women who havehad a spontaneous preterm birth to be evaluated and compared to controlsin order to identify biomarkers that can be used to predict pretermdelivery. Moreover, the vaginal mucosa of pregnant females may beanalysed using the desorption electrospray ionisation (“DESI”) massspectrometry based technique disclosed herein to analyse, e.g., diagnoseor predict the risk of, a (spontaneous) preterm birth.

Mass spectral profiling of vaginal mucosa can enable an earlyidentification of females, e.g., women who are at risk of infectionduring pregnancy based upon microbial, e.g., bacterial diversity in thevaginal mucosa. Furthermore, this enables targeted treatment responsestrategies.

Various embodiments are contemplated and include: (i) identification ofvaginal mucosa metabolite biomarkers that are related to specificmicrobial, e.g., bacterial communities, optionally as determined usingsequencing microbiome analysis; (ii) profiling of vaginal mucosalmembrane during healthy pregnancy wherein microbe, e.g.,bacteria-specific metabolites and signatures that are excreted duringhealthy pregnancy may be characterised in detail; and (iii)identification of diagnostic and prognostic metabolic signatures fromvaginal mucosa membranes with poor pregnancy outcomes (e.g. pretermdelivery).

FIG. 14A shows desorption electrospray ionisation (“DESI”) massspectrometry analysis of a bacteria (Klebsiella pneumonia) sample on aswab in accordance with an embodiment. The data illustrated in FIG. 14Ashows that bacterial samples can be detected using desorptionelectrospray ionisation (“DESI”) mass spectrometry on swabs, accordingto various embodiments. FIG. 14B shows for comparison rapid evaporativeionisation mass spectrometry (“REIMS”) time of flight (“TOF”) massspectrometry data of a corresponding bacterial sample measured directlyfrom an agar plate. The peaks highlighted by stars were detected withboth ionisation techniques.

Desorption electrospray ionisation (“DESI”) swab analysis formicroorganism detection was further tested on six cultivated speciesincluding Candida albicans, Pseudomonas montelli, Staphylococcusepidermis, Moraxella catarrhalis, Klebsiella pneumonia and Lactobacillussp. These are all important bacteria and fungi species that wereisolated from vaginal mucosal membranes of pregnant patients and whichwere identified by sequence analysis such as 16S rRNA gene sequencing.

A swab was quickly dipped into a solution of diluted biomass from eachspecies in 10 μL methanol, followed by desorption electrosprayionisation (“DESI”) mass spectrometry analysis of the swab surface.

FIGS. 15A-C show microorganism analysis using desorption electrosprayionisation (“DESI”) mass spectrometry on swabs.

FIG. 15A shows averaged desorption electrospray ionisation (“DESI”) massspectra of diverse analysed microorganism species including Candidaalbicans, Pseudomonas montelli, Staphylococcus epidermis, Moraxellacatarrhalis, Klebsiella pneumonia and Lactobacillus sp.

FIGS. 15B and 15C show PCA plots showing a separation between thevaginal mucosa (pregnant and non-pregnant group) and the microorganismspecies within the first two components. In addition, a separation canbe observed between the different bacteria and fungi species.

Unique spectral features were observed in the mass spectra as shown inFIG. 15A resulting in the ability to separate between differentmicroorganism classes as well as from the vaginal mucosa in the PCAscore plots (FIGS. 15B and 15C) within the first two components.

This result shows the potential to characterise microbe, e.g.,bacteria-specific and host-response metabolite biomarkers and signaturesfrom specific microbial, e.g., bacterial communities from the animal,e.g., human mucosal membrane using desorption electrospray ionisation(“DESI”) mass spectrometry on medical swabs.

It will be appreciated that various embodiments provide a new desorptionelectrospray ionisation (“DESI”) mass spectrometry setup fornon-invasive and fast analysis of the mucosal metabolome profile fromthe surface of medical swabs. This arrangement has been successfullyshown to be capable of differentiating animal, e.g., human mucosalmembrane models and to enable microorganism identification. The methodis capable of readily distinguishing different mucosal sites,biochemical alterations induced by physiological events such aspregnancy, and permits rapid identification of intact bacterial andfungal species.

Since desorption electrospray ionisation (“DESI”) mass spectrometryanalysis causes minimal sample destruction to the majority of the samplesurface material, according to various embodiments the medical swab canoptionally be sent directly after desorption electrospray ionisation(“DESI”) analysis, e.g. to a microbiological lab, for further assessmentsuch as cultivation, microbe identification/confirmation, and/or nextgeneration sequencing analysis. As the resultant desorption electrosprayionisation mass spectrometry spectral profiles according to variousembodiments harbour information descriptive of mucosal biochemistry aswell as microbal-host interactions, the method according to variousembodiments is applicable to a wide range of clinical applications.

Various embodiments provide a new point of care mucosal screeningdiagnostic method which uses standard cotton medical swabs as both thesampling probe for mucosal membrane uptake and ionisation probe fordesorption electrospray ionisation (“DESI”) mass spectrometry analysis.After data acquisition the obtained spectra may be compared with spectracollected in a database to provide a rapid diagnosis to the patient,e.g., within several seconds.

Various embodiments relate to the application of the desorptionelectrospray ionisation (“DESI”) technique for direct metabolomicprofiling of specific mucus models (nasal, vaginal, pharyngeal,bronchial, oesophageal) from the surface of standard medical swabs.Various embodiments relate to a rapid point-of-care diagnostic methodfor diseases, optionally selected from any of the diseases mentionedherein, e.g., inflammatory and pathogen-related diseases such as inimmunological disorders, dysbiosis in the microflora (which may, e.g. beindicative of the risk of pre-term delivery during pregnancy),microbial, e.g., bacterial infections, or the detection of cancer orpre-cancerous states. The metabolomic profiling of animal, e.g., humanmucosal membrane followed by detailed statistical analysis permits theidentification of disease-specific metabolic profiles and/or taxonspecific microbial, e.g., bacterial markers in a rapid, robust mannerconducive to a point-of-care diagnostic method.

As shown in FIG. 16, according to various embodiments, desorptionelectrospray ionisation (“DESI”) mass spectral analysis 160 of a samplesampled 161 onto a swab may be subjected to statistical analysis 162 inorder to provide a diagnosis 163 (or prognosis).

The sample may be additionally or alternatively be analysed by rapidevaporative ionisation mass spectrometry (“REIMS”) mass spectrometry164.

Embodiments are contemplated wherein multiple different analysistechniques may be applied to the same swab (or another swab) so as toadditionally perform analyses that rely on culturing 165, such as DNAextraction and PCR analysis, e.g., to produce complementary 16S rRNAmicrobiome data.

As shown in FIG. 16, any one or more or all of the additional analysesmay be used to validate the desorption electrospray ionisation (“DESI”)based diagnosis 163.

FIG. 17A illustrates how continuous rotation of a swab whilst subjectingthe swab to desorption electrospray ionisation (“DESI”) analysis canresult in an enhanced signal intensity.

Rapid Evaporative Ionisation Mass Spectrometry (“REIMS”) Analysis of aSwab

Various embodiments described herein also relate to methods of rapidevaporative ionisation mass spectrometry (“REIMS”) analysis of a swab,wherein a sample on a swab is subjected to rapid evaporative ionisationmass spectrometry (“REIMS”) analysis. This approach, however, isdestructive for the swab, and in the bipolar mode the contact closure ofthe electrodes is restricted.

When a swab is analysed by rapid evaporative ionisation massspectrometry, then the swab may be dipped, soaked or otherwise immersedin a fluid (such as water) prior to be being subjected to rapidevaporative ionisation mass spectrometry (“REIMS”) analysis.

As also illustrated in FIG. 17B, soaking a swab in a fluid prior torapid evaporative ionisation mass spectrometry has also been shown toenhance the signal intensity.

The rapid evaporative ionisation mass spectrometry (“REIMS”) techniquewill now be described in more detail with reference to FIG. 18.

FIG. 18 illustrates a method of rapid evaporative ionisation massspectrometry (“REIMS”) wherein bipolar forceps 1 may be brought intocontact with in vivo tissue 2 of a patient 3. In the example shown inFIG. 18, the bipolar forceps 1 may be brought into contact with braintissue 2 of a patient 3 during the course of a surgical operation on thepatient's brain. However, according to various embodiments, and as shownin FIG. 17B, the bipolar forceps 1 may be brought into contact with asample provided on a medical swab.

An RF voltage from an RF voltage generator 4 may be applied to thebipolar forceps 1 which causes localised Joule or diathermy heating ofthe tissue 2 or sample. As a result, an aerosol or surgical plume 5 isgenerated. The aerosol or surgical plume 5 may then be captured orotherwise aspirated through an irrigation port of the bipolar forceps 1.The irrigation port of the bipolar forceps 1 is therefore reutilised asan aspiration port. The aerosol or surgical plume 5 may then be passedfrom the irrigation (aspiration) port of the bipolar forceps 1 to tubing6 (e.g. ⅛″ or 3.2 mm diameter Teflon® tubing). The tubing 6 is arrangedto transfer the aerosol or surgical plume 5 to an atmospheric pressureinterface 7 of a mass spectrometer 8 and/or ion mobility analyser.

According to various embodiments a matrix comprising an organic solventsuch as isopropanol may be added to the aerosol or surgical plume 5 atthe atmospheric pressure interface 7. The mixture of aerosol 3 andorganic solvent may then be arranged to impact upon a collision surfacewithin a vacuum chamber of the mass and/or ion mobility spectrometer 8.According to one embodiment the collision surface may be heated. Theaerosol is caused to ionise upon impacting the collision surfaceresulting in the generation of analyte ions. The ionisation efficiencyof generating the analyte ions may be improved by the addition of theorganic solvent. However, the addition of an organic solvent is notessential.

Analyte ions which are generated by causing the aerosol, smoke or vapour5 to impact upon the collision surface are then passed throughsubsequent stages of the mass spectrometer (and/or ion mobilityanalyser) and are subjected to analysis such as mass analysis and/or ionmobility analysis in a mass analyser or filter and/or ion mobilityanalyser. The mass analyser or filter may, for example, comprise aquadrupole mass analyser or a Time of Flight mass analyser.

Modified Swabs

Various further embodiments are directed to a modified, chemicallyfunctionalised and/or solid-phase microextraction (“SPME”) swabapproach.

As discussed above, various chemically modified swabs for use withdesorption electrospray ionisation (“DESI”) mass spectrometry inaccordance with various embodiments have been found to exhibit animproved sensitivity and/or reduced background compared withconventional (non-modified) swabs. Modified swabs as described in moredetail below exhibit an improved signal to noise ratio compared withconventional (non-modified) swabs across particular mass to charge ratioranges.

As illustrated in FIG. 19, standard cotton swabs are commerciallyavailable and are relatively non-invasive. However, swabs comprisefibrous material having absorbing properties, the swabs releasemolecules relatively poorly, provide non-selective extraction and canresult in a relatively high background signal being observed.

In contrast, coated or chemically modified swabs according to variousembodiments can beneficially provide a solid surface, enable selectiveextraction and exhibit improved sensitivity.

According to various embodiments, a (standard) medical swab may bewetted or otherwise functionalised with one or more sorbents in ordereither: (i) to increase the intensity or signal of observed analyte ionsacross one or more particular mass to charge ratio ranges; and/or (ii)to reduce the intensity or signal due to undesired background ionsacross one or more particular mass to charge ratio ranges.

In accordance with various embodiments, standard cotton medical swabsmay be coated with ODS/C18 (octadecyl), polydimethylsiloxane (“PDMS”),Oasis® MAX (mixed-mode cation exchange), Oasis® HLB(hydrophilic-lipophilic-balanced) and/or Oasis® MCX (mixed-mode cationexchange). For example, according to an embodiment the swab may beprovided with a monolayer or multiple layers of sorbent materials suchas C18 (octadecyl), C18 (octadecyl) EC (end capped), HLB (hydrophiliclipophilic balanced particles) and/or divinyl benzene (“DVB”). Thesevarious sorbent materials may be used to enhance the extractionefficiency of certain compounds from the mucosal membrane matrix.

FIG. 20 illustrates the improved sensitivity that is provided usingmodified swabs, in accordance with various embodiments. Desorptionelectrospray ionisation (“DESI”) mass spectrometry analysis of nasalfluid on a swab was performed using a standard cotton swab (FIG. 20A), aswab modified with ODS/C18 (FIG. 20B), and a swab modified with Oasis®HLB (FIG. 20C). In particular, an improved lipid signal is observed overthe mass to charge ratio range of around m/z 730-890.

FIG. 21 shows a collection of different coated materials comprisingdifferent formats, shapes and sorbent materials.

The biocompatible and functionalised polybutylene terephthalate (“PBT”)plastic, swabs and fibres may be prepared by dip-coating the materialsurface, e.g. using peroxyacetyl nitrate (“PAN”) as an adhesive for thesorbent materials. Using the dip-coating technique a mono-layer coatingcan be achieved with a coating thickness of approximately 5-10 μm.

Functionalised swabs with C18 (octadecyl), C18 (octadecyl) EC(endcapped), DVB (divinyl benzene) WAX (weak anion exchange) and HLB(hydrophilic-lipophilic-balanced) coating were tested for desorptionelectrospray ionisation (“DESI”) mass spectrometry metabolic profilingof saliva and compared with the standard medical cotton desorptionelectrospray ionisation (“DESI”) mass spectrometry analysis approach.

As shown in FIG. 22, once functionalised, a swab may require washing 210and/or conditioning 211 prior to use. Washing 210 and/or conditioning211 steps remove any contaminants left over from the manufacturingprocess which might interfere with the desorption electrosprayionisation (“DESI”) mass spectrometry analysis. For example, washing 210may involve soaking in a first solvent to remove any contaminants leftover from the manufacturing process whilst conditioning 211 may involvesoaking in a second solvent to remove any remaining contaminants,including any unwanted residue of the first solvent. Suitable solventsfor these steps include methanol, acetonitrile (ACN), isopropanol, waterand mixtures thereof.

Once the swab has been contacted with the sample or bodily surface 212,it may be washed 214 to remove unbound material which may interfere withthe analysis, leaving the analyte of interest bound to the swab. Forexample, if the analyte of interest is a lipid, unbound salts or polarmolecules can be removed by washing 214. The swabs are then analysed 215by desorption electrospray ionisation (“DESI”) mass spectrometry or avariant thereof such as using a desorption electroflow focusingionisation (“DEFFI”) ion source.

An optimised workflow was designed in which swabs were washed 210 inMeOH/ACN/(CH₃)₂CHOH (50:25:25) for around 1 hour, conditioned 211 inMeOH:H₂O (50:50) for around 1 minute, dipped in analyte (e.g. saliva)solution for around 2 min and 30 minutes so as to sample 212 theanalyte, dried 213 for around 5 minutes, and rinsed 214 by dipping inwater for around 1 second, prior to desorption electrospray ionisation(“DESI”) mass spectrometry analysis 215 with MeOH:H₂O (95:5). Thisworkflow was shown to enhance the extraction efficiency and sampleclean-up during mucosal membrane analysis.

FIGS. 23A-E show a comparison of saliva spectra obtained in negative(left) and positive (right) ion mode obtained using a standard medicalswab (FIG. 23A) and three layer coated swabs (FIGS. 23B-E) which werecoated with four different sorbents (C18 (octadecyl), C18 (octadecyl) EC(end capped), HLB (hydrophilic-lipophilic-balanced) and DVB (divinylbenzene) WAX (weak anion exchange)).

FIG. 23A shows a saliva spectra obtained in negative ion mode (left) andpositive ion mode (right) using a standard medical swab, FIG. 23B showsa saliva spectra obtained in negative ion mode (left) and positive ionmode (right) using a standard medical swab coated with three layers ofC18 (octadecyl) sorbent, FIG. 23C shows a saliva spectra obtained innegative ion mode (left) and positive ion mode (right) using a standardmedical swab coated with three layers of C18 (octadecyl) EC (end capped)sorbent, FIG. 23D shows a saliva spectra obtained in negative ion mode(left) and positive ion mode (right) using a standard medical swabcoated with three layers of HLB (hydrophilic-lipophilic-balanced)sorbent and FIG. 23E shows a saliva spectra obtained in negative ionmode (left) and positive ion mode (right) using a standard medical swabcoated with three layers of DVB (divinyl benzene) WAX (weak anionexchange) sorbent.

Whereas medical cotton swabs show high background peaks especially inpositive ion mode (highlighted peaks), coated swabs results in cleanerbackground spectra and enhanced lipid sensitivity from the saliva matrix(green highlighted) using C18 (octadecyl) in negative ion mode (m/z700-900) and HLB (hydrophilic-lipophilic-balanced) and DVB (divinylbenzene) WAX (weak anion exchange) swabs in positive ion mode (m/z600-720).

In summary, functionalized swabs were found to improve sensitivity forhydrophobic analytes, due to improved selective extraction efficiency ofunpolar analytes from the saliva matrix compared to standard cottonswabs.

Various embodiments described herein provide an optimised desorptionelectrospray ionisation (“DESI”) mass spectrometry method for metabolicprofiling of mucosal membrane samples on medical swabs.

Various embodiments facilitate the differentiation between differentmucosa models.

Various embodiments allow bacterial metabolic profiles to be obtainedfrom swabs.

Furthermore, functionalised swabs in accordance with various embodimentsimprove the sensitivity, e.g. for hydrophobic analytes.

According to various embodiments, rapid evaporative ionisation massspectrometry (“REIMS”) may be used as a complementary analysis techniquefor mucosal profiling.

It will be appreciated that next generation sequencing techniques suchas 16S RNA sequencing permit identification and characterization ofbacteria that are colonized in the human mucosal membrane. However,clinical implementation for bacterial identification is limited due tocost and time constrains of this approach. Desorption electrosprayionisation (“DESI”) mass spectrometry analysis of mucosal swabs,however, fulfils all criteria set for a routine diagnostic procedure.Using desorption electrospray ionisation (“DESI”) mass spectrometry torapidly and directly identify metabolite signatures excreted in animal,e.g., human mucosal membrane by specific microbes, e.g., bacteriaenables objective biochemical information to be generated which enablesmicrobes, e.g., bacteria to be identified and which enhances currentclinical decisions making (e.g. target antibiotic treatment) in thecontext of analysis, e.g., diagnosis of diseases, such as any of thediseases mentioned elsewhere herein, e.g., infections, dysbiosis,cancer, and/or inflammatory disease.

Modified Swab Surface Chemistry

Various embodiments provide a medical swab for use in the methods ofvarious embodiments, wherein the swab has been chemically modified toenhance selectivity for an analyte.

Medical swabs normally comprise a head portion, often referred to as abud, and a shaft which may be attached to or integral with the head. Theshaft may be formed from plastic, wood, rolled paper or wire. The headportion is often hydrophilic. The head portion may be formed fromcotton, rayon, plastic fibres or foams. Suitable plastics for both theshaft and head include polyurethanes and polyesters such as polyethyleneterephthalate (PET) and polybutylene terephthalate (PBT).

References herein to cotton, rayon, plastic fibres or foam swabs arereferences to the materials from which the head of the swabs is made.

In principle, any known medical swabs or medical sampling devices may bechemically modified (functionalised) to improve their selectivity forparticular analytes of interest prior to analysis using desorptionelectrospray ionisation (“DESI”) mass spectrometry, or a variantthereof. The swabs may be disposable (i.e. intended for a single use).Swabs for chemical modification/functionalization include cotton, rayon,polyester and foam swabs, particularly cotton or polyester swabs.

Chemical modification of a swab involves introduction of a suitablefunctionalising chemistry to the surface of the swab. Thefunctionalising chemistry may be attached to the swab by chemical means,for example via covalent bonds, or by physical means, for example viaphysical entrapment or by using a suitable adhesive material. In variousembodiments, the chemical modification involves formation of a coatingon the surface of the swab.

The nature of the surface functionalization will depend on the type ofanalyte of interest. After functionalization, the surface of the swabmay be hydrophilic or hydrophobic. It may also comprise ionic groups.For example, hydrophobic or lipophilic surfaces are beneficial foranalysis of lipids, whilst charged or hydrophilic surfaces may bebeneficial for the analysis of proteins or certain drug metabolites.

The chemical modification should preferentially bind the analyte oranalytes of interest, but should not bind the analyte so tightly thatthe analyte cannot subsequently be removed for analysis by massspectrometry.

Functionalised swabs may be used to analyse a sample, such as urine,after the sample have been removed from a patient. The surfacefunctionalization used in swabs for such in vitro use need not bebiocompatible. Alternatively, functionalised swabs may be used directlyto collect a sample from the patient, for example by swabbing a mucosalmembrane. Swabs which are intended for in vivo use or to come intodirect contact with a patient should be biocompatible.

The surface functionalization may be introduced to the swab throughadsorption or absorption and though processes including, but not limitedto: solution phase processes, gas phase processes, chemical vapourdeposition, molecular vapour deposition, atomic layer deposition, dipcoating, electrochemical coating, or spray coating.

Direct chemical attachment of a functionalising molecule to the swab mayoccur by reaction of a functional group in the functionalising moleculewith a functional group in the swab material to form a covalent bond.Covalent attachment of the functionalising molecule to the swab caninclude, but is not limited to, reaction of a functional group of theswab such as an alcohol, aldehyde, amine, carboxylic acid or alkenegroup to form a silyl-ether, ether, thio-ether, carbamate, carbonate,carbon-carbon bon, carbon-nitrogen bond, urea or ester. For example,cellulosic swabs such as cotton or rayon swabs will comprise a pluralityof hydroxy groups which may react with functional groups such ascarboxylic acids in the functionalising molecule, leading to covalentattachment of the functionaliser to the surface of the swab via esterlinkages.

Methods for functionalising cellulosic surfaces are known in the art andinclude those disclosed in US2009/0126891, the content of which isincorporated herein by reference.

Alternatively, the chemical modification of a swab may involve physicalentrapment of a suitable functionaliser by the swab. This method may beuseful when the functionaliser is a solid particle such as afunctionalised silica particle or an ion exchange resin particle.

Another alternative is to use a thin layer of adhesive or other bindermaterial to attach functionalised particles to the surface of a swab.Any known medical grade adhesive may be potentially used, includingcyanoacrylates, epoxy adhesives and acrylate adhesives. Suitable suchadhesives include the Loctite® medical grade adhesives available fromHenkel Corporation, Connecticut, USA. Any inert, biocompatible particlesmay potentially be used including silica, hybrid silica-organic orcarbon particles. Suitable particles are generally about 1 to about 60micrometers in diameter, and may be about 2 to about 10 micrometers indiameter, and may be about 2 to about 5 micrometers in diameter.

A beneficial surface functionalization of a swab involves attachment ofsolid-phase extraction materials to the surface of the swab, for exampleby physical entrapment or through use of a suitable adhesive or bindermaterial. As used herein, the term “solid phase extraction materials”refers to solid materials useful as stationary phases in gas or liquidchromatography. Such materials are usually in particulate form and areoften silica or polymer resin based. Solid-phase extraction materialsfor the analysis of lipids may be reverse-phase stationary phases.

Functionalised silica and hybrid silica/organic particles are well knownfor use as stationary phases in liquid chromatography. Methods offunctionalising silica surfaces to render them more selective foranalytes of interest are known in the art. Suitable modificationsinclude those disclosed in US 2012/0141789 and US 2008/0073512, thedisclosures of which are incorporated herein by reference.

Surface modifiers for chromatographic stationary phases typicallyinclude organic functional groups which impart a certain chromatographicfunctionality to the stationary phase. Functional groups on the surfaceof the stationary phase particles may be derivatized by reaction withsuitable modifiers. Silica particles possess silanol groups whilstsilica/organic hybrid particles may possess both organic groups andsilanol groups which may be derivatized.

Suitable surface modifiers for chromatographic stationary phases includethose having the formula Z_(a)(R′)_(b)Si—R², where Z═Cl, Br, I, C₁-C₅alkoxy, dialkylamino or trifluoromethanesulfonate; a and b are each aninteger from 0 to 3 provided that a+b=3; R¹ is a C₁-C₆ straight, cyclicor branched alkyl group, and R² is a functionalizing group.

R′ may be selected from the group consisting of methyl, ethyl, propyl,isopropyl, butyl, t-butyl, sec-butyl, pentyl, isopentyl, hexyl andcyclohexyl.

The functionalizing group R² may include alkyl, alkenyl, alkynyl, aryl,cyano, amino, diol, nitro, ester, cation or anion exchange groups, analkyl or aryl group containing an embedded polar functionalities orchiral moieties. Examples of suitable R² functionalizing groups includechiral moieties; C₁-C₃₀ alkyl, including C₁-C₂₀, such as octyl (C₈),octadecyl (C18) and triacontyl (C₃₀); alkaryl, e.g., C₁-C₄-phenyl;cyanoalkyl groups, e.g., cyanopropyl; diol groups, e.g., propyldiol;amino groups, e.g., aminopropyl; and alkyl or aryl groups with embeddedpolar functionalities, e.g., carbamate functionalities such as disclosedin U.S. Pat. No. 5,374,755; and chiral moieties. Such groups includethose of the general formula:

wherein 1, m, o, r and s are 0 or 1, n is 0, 1, 2 or 3 p is 0, 1, 2, 3or 4 and q is an integer from 0 to 19; R3 is selected from the groupconsisting of hydrogen, alkyl, cyano and phenyl; and Z, R′, a and b aredefined as above. The carbamate functionality may have the generalstructure indicated below:

wherein R⁵ may be, e.g., cyanoalkyl, t-butyl, butyl, octyl, dodecyl,tetradecyl, octadecyl, or benzyl. Advantageously, R⁵ is octyl, dodecyl,or octadecyl.

R² may be a C₁-C₃₀ alkyl group, and may be a C₁-C₂₀ alkyl group.

Particularly beneficial surface modifiers are selected from the groupconsisting of octyltrichlorosilane, octadecyltrichlorosilane,octyldimethylchlorosilane and octadecyldimethylchlorosilane, and may bein particular octyltrichlorosilane or octadecyltrichlorosilane.

Particulate solid phase extraction materials are available from WatersCorporation, USA. Particularly suitable such materials are silicaparticles functionalised with octadecyl (C18) groups orpolydimethylsiloxane (PDMS) groups.

Alternatively, swabs may be functionalised by attachment of particulatepolymer resins to the surface of the swab, for example by physicalentrapment or through use of a suitable adhesive or binder. Suitablesuch resins are known in the art as stationary phases for use inchromatography. These include reversed-phase, ion exchange, and mixedmode stationary phases, including ion exchange-reverse phase stationaryphases.

Reverse-phase chromatography stationary phases may be particularlysuitable for preparing swabs for analysis of lipids. Suitablereversed-phase stationary phases include polydivinylbenzene (DVB) andcopolymers of N-vinylpyrrolidone and divinylbenzene, such as Oasis® HLBavailable from Waters Corporation.

Mixed-mode ion exchange/reversed-phase sorbents based on modified Nvinylpyrrolidone/divinylbenzene copolymers may also be used. Suchcopolymers in which some of the benzene rings are sulfonated orcarboxylated can provide cation-exchange functionality. Such copolymersmodified by attachment of imidazolium, —CH₂-piperazine groups orquaternary ammonium groups such as —CH₂N⁺(CH₃)₂(C₄H₉) to some of thebenzene rings can provide anion exchange functionality. Suitable suchsorbents are available from Waters Corporation under the trade namesOasis® MCX, Oasis® WCX, Oasis® MAX and Oasis® WAX.

Solid particles for attachment to swabs may also be functionalised withpolymer coatings. Polymer coatings are known in the literature and maybe provided generally by polymerization or polycondensation ofphysisorbed monomers onto the surface of the particle without chemicalbonding of the polymer layer to the support (type I), polymerization orpolycondensation of physisorbed monomers onto the surface with chemicalbonding of the polymer layer to the support (type II), immobilization ofphysisorbed prepolymers to the support (type III) and chemisorption ofpresynthesized polymers onto the surface of the support (type IV): see,e.g., Hanson, et al., J. Chromat. A656 (1993) 369-380, the text of whichis incorporated herein by reference.

Any polymers used to make solid phase extraction materials maypotentially be used to form polymeric coatings on the surface of a swab.Suitable coating polymers include polydivinylbenzene (DVB), copolymersof N-vinylpyrrolidone and divinylbenzene, and polydimethylsiloxane.

Coating a particle with a polymer may be used in conjunction with othersurface modifications as described above. In particular, SPEM particlesmay be embedded in the polymer coating such that the coating acts as abinder to trap the particles on or near the surface of the swab.

Functionalised swabs may also be prepared by forming a solution ordispersion of the chemical modifier and dipping a swab into the solutionor dispersion. The swab is then allowed to dry and the dipping steprepeated, as necessary. Such dipping may allow the formation ofmonolayers of the chemical modifier on the surface of the swab. Thethickness of a layer formed by this method may be in the region of 5-10micrometers.

Fibres for use as solid phase microextraction (SPME) media are known inthe art. Such fibres normally comprise a fused silica fibre or metalwire with a thin polymer coating. For example, C18 coated fused-silicafibres have been proposed for use in sampling for drugs in urine(Kennedy et al, Analytical Chemistry, Vol. 82, No. 17, 1 September2010). Swabs for use in the various embodiments may further comprisesuch fibres.

In various embodiments, the swabs do not include any fused silica-basedfibers or any metal fibers/wires.

Sterilisation of Swabs

If the functionalised swabs are to be used to analyse a sample which hasbeen removed from a patient, then the swabs are not necessarily requiredto be sterilised prior to use. For example, swabs used to analyse urinesamples may not require sterilisation prior to use. However, any swabswhich are intended to come into contact with a patient will requiresterilisation prior to use. Any standard technique in the art known forsterilising medical swabs may potentially be used to sterilise thefunctionalised swabs of various embodiments. Suitable sterilisationmethods include, without limitation, autoclaving, heating, gammaradiation and ethylene oxide sterilisation.

Multiple Analyses of the Same Swab

As discussed above, a particular benefit of using desorptionelectrospray ionisation (“DESI”) mass spectrometry to analyse a sampleprovided on a medical swab is that multiple different analyses of thesame sample, i.e. of the same swab, may be performed.

Performing multiple different analyses of or on the same sample enablesmultiple different sets of information about the same sample to beobtained in a particularly convenient and efficient manner. This is inparticular possible because desorption electrospray ionisation (“DESI”)mass spectrometry is a relatively non-destructive analysis technique andalso because various commercial analysis techniques, such as culturingtechniques and nucleic acid sequencing techniques, e.g., 16S rRNAsequencing techniques, are optimised to use samples which are providedon medical swabs.

Accordingly, following a single sample acquisition onto a swab, thesample on the swab may be analysed multiple times using multipledifferent analysis techniques, where at least one of the techniques(e.g. the first technique used) comprises desorption electrosprayionisation (“DESI”) mass spectrometry.

According to various other embodiments, data directed analyses of asample on a swab (the same sample on the same swab) may be performed.For example, depending upon the results of the first (e.g. desorptionelectrospray ionisation (“DESI”) mass spectrometry) analysis and/orwhere an ion of interest is detected using the first (e.g. desorptionelectrospray ionisation (“DESI”) mass spectrometry) analysis, a furtheranalysis may be selected, altered and/or optimised and performed.

In these embodiments, one or more of the analysis techniques maycomprise a culturing analysis method, e.g. where the swab is contactedwith, e.g., wiped across or dipped into, a solid or liquid culturingmedium, the culturing medium is incubated, and then the culturing mediumis examined, e.g., under a microscope to identify any microbes present.

One or more of the analysis techniques may comprise a gene sequencingmethod such as a 16S rRNA sequencing method for identifying microbes.

One or more of the analysis techniques may comprise a Matrix-AssistedLaser Desorption Ionisation (“MALDI”) method for identifying microbes.

One or more of the analysis techniques may comprise a Rapid EvaporativeIonisation Mass Spectrometry (“REIMS”) method for identifying microbes.

As shown and described above in relation to FIG. 16, any one or more orall of the additional analyses may be used to validate and/or supplementa desorption electrospray ionisation (“DESI”)based identification ordiagnosis.

Analysis of Swabs Comprising a Faecal or Body Fluid Specimen UsingAmbient Ionisation Mass Spectrometry, e.g. DESI and/or REIMS Technology

Analysis of a faecal or body fluid specimen according to variousembodiments may provide information about a disease and/or microbiome,optionally a mucosal microbiome and/or the microbiome of the GI lumen.Thus, optionally, the method may involve the analysis of a swabcomprising a faecal and/or body fluid specimen. For example, a faecaland/or body fluid specimen may be analysed for the presence of a cell, acompound, and/or a microbe.

The method may optionally allow an analysis of metabolic differencesbetween various conditions, which may optionally be selected from any ofthe conditions listed elsewhere herein, e.g., Irritable Bowel Syndrome,Colorectal cancer and/or Inflammatory Bowel Disease. By identifyingtaxonomic specific biomarkers the method may optionally allow theanalysis, e.g., diagnosis, of microbial infections and/or mixedmicrobial communities.

The cell may, e.g., be a mammalian cell, a white blood cell, a red bloodcell, a foetal cell, and/or a cancer cell.

Optionally, a faecal and/or body fluid specimen may be analysed for thepresence of a microbe and/or to analyse a microbiome. Details ofanalysis of microbes and/or the microbiome are provided elsewhereherein.

Optionally, a faecal and/or body fluid specimen may be analysed for thepresence of a compound. The compound may, e.g., comprise or consist of abiomolecule, an organic compound, and/or an inorganic compound. It mayoptionally be selected from any of the compounds listed elsewhereherein. Optionally, it may be bile, haemoglobin, or a derivative of anythereof.

Optionally, a faecal and/or body fluid specimen other than blood may beanalysed for the presence of blood. For example, the presence of bloodin urine may be indicative of an infection or other disease. Forexample, the presence of blood in a faecal specimen may optionally beused to analyse a bleed in the GI tract and/or anus. Optionally, thebleed may be indicative of a disease selected, for example, from analfissure, diverticular disease, a polyp, an inflammatory disease,angiodysplasia, and/or any of the diseases mentioned elsewhere herein.

Optionally, a faecal and/or body fluid specimen may be analysed for thepresence of bile or a derivative thereof, e.g., to analyse a liverand/or kidney disease, and/or any of the diseases mentioned elsewhereherein.

The analysis of faecal specimens may optionally involve the useforceps-based rapid evaporative ionisation mass spectrometry (“REIMS”),wherein a sample of the faecal specimen may be taken between the forcepsand the probes may then be drawn together.

FIG. 24 shows a spectrum observed when analysing stool samples using therapid evaporative ionisation mass spectrometry (“REIMS”) technique.

Desorption Electrospray Ionisation (“DESI”) Sprayer with Heated TransferCapillary

FIG. 25A shows an embodiment and comprises a Desorption ElectrosprayIonisation (“DESI”) sprayer 300 in which a solvent capillary 302 may bearranged to direct electrically charged droplets 304 of solvent at aswab surface 310. A sample 311 may be located on the swab surface 310,which may comprise analyte particles. The charging of the solventdroplets may be achieved through the use of a high-voltage power supply306 that contacts the capillary 302. The high-voltage power supply 306may comprise an electrode 307 which may contact any portion of thecapillary 302 so that it is operable to charge the solvent droplets asthey leave an outlet end 303 of the capillary 302. The outlet end 303 ofthe capillary may be directed towards the swab surface 310.

A sheath gas 308 (e.g., nitrogen) may be arranged to surround thecapillary 302 so as to nebulise the solvent as it emerges from thecapillary 302 and direct the electrically charged solvent droplets 304towards the swab surface 310. The sheath gas may be introduced through atube 312 that may be coaxial to the solvent capillary 302, having aninlet 314 at an end distal to the swab surface 310 and an outlet 316 atan end facing the swab surface 310.

The outlet 316 of the sheath gas tube 312 may be concentric to theoutlet end 303 of the capillary, which can facilitate in nebulising thesolvent as it emerges from the capillary 302. The solvent emerging fromthe outlet end 303 of the solvent capillary 302 may be nebulised by thesheath gas 308. A connector 318 may connect the tube 312 to a source ofgas suitable to use as a sheath gas. The sheath gas 308 may comprisenitrogen or standard medical air, and the source of sheath gas may be asource of nitrogen gas or standard medical air.

As the solvent droplets 304 contact the swab, analyte particles on theswab can desorb and the charged droplets and analyte mixture 320 may betransferred into a transfer capillary or transfer device 330 that maylead to an ion analyser, mass analyser or filter and/or ion mobilityanalyser and/or mass spectrometer 340. The charged droplet and analytemixture may be transferred through an inlet 332 of the transfercapillary or transfer device 330. This may be achieved by placing theopposite end 333 of the transfer capillary or transfer device 330 in alow pressure region 352, for example a vacuum stage of the mass analyseror filter and/or ion mobility analyser and/or mass spectrometer 340.

The charged droplet and analyte mixture (including e.g., analyte ions)may be transferred by ion optics 352 to an analysis region of the ionanalyser and/or ion mobility analyser and/or mass spectrometer 340. Theion optics 352 may comprise an ion guide, for example a StepWave® ionguide.

The analyte ions may be guided to the analysis region by applyingvoltages to the ion optics 352. The analyte ions may then be analysed bythe mass analyser or filter and/or ion mobility analyser and/or massspectrometer 340.

According to an embodiment the ion analyser and/or mass analyser orfilter and/or ion mobility analyser and/or mass spectrometer 340 maycomprise an ion mobility spectrometer. According to a yet furtherembodiment the ion analyser and/or mass analyser or filter and/or ionmobility analyser and/or mass spectrometer 340 may comprise thecombination of an ion mobility spectrometer and a mass spectrometer.

As a result of the analysis, chemical information about the sample 311may be obtained.

One or more heaters may be provided to heat the various parts of theapparatus shown in FIG. 25A. For example, a heater may be provided toheat one or more of the solvent capillary 302, the sheath gas tube 312,the swab surface 310 and the transfer or inlet capillary 330.

The one or more heaters may comprise a wire heater (e.g., a tungstenwrap) and/or may be configured to heat the respective part to at least50° C., 100° C., 200° C., 300° C., 400° C., 500° C., 600° C., 700° C. or800° C. However, any type of heater may be used that has the function ofheating the respective part, for example a blower or an inductiveheater.

FIG. 25A shows a first heater 342 that may be arranged and adapted toheat the transfer or inlet capillary 330, such that the solvent andanalyte mixture 320 may be heated before being passed onward, forexample to the mass analyser or filter and/or ion mobility analyserand/or mass spectrometer 340.

The first heater 342 may be located anywhere along the solvent capillary330, for example adjacent to or at the inlet 341 of the ion analyser,mass analyser or filter and/or ion mobility analyser and/or massspectrometer. Alternatively, the first heater 342 may be locatedadjacent to or at the inlet 332 of the solvent capillary or transferdevice 330. The first heater 342 may comprise a wire heater (e.g., atungsten wrap) and/or may be configured to heat the inlet capillary toat least 50° C., 100° C., 200° C., 300° C., 400° C., 500° C., 600° C.,700° C. or 800° C.

A second heater 344 may be arranged and adapted to heat the sheath gastube 312, such that the solvent and/or sheath gas may be heated.

The second heater 344 may be located at the end of the tube 312 nearestthe swab surface 310, such that the solvent and/or sheath gas may beheated before being directed at the swab surface 310. The second heater344 may comprise a wire heater (e.g., a tungsten wrap) and/or may beconfigured to heat the tube 312 and/or the solvent and/or the sheath gasto at least 50° C., 100° C., 200° C., 300° C., 400° C., 500° C., 600°C., 700° C. or 800° C.

A third heater 346 may be arranged and adapted to heat the solventcapillary 302, such that the solvent may be heated.

The third heater 346 may be located anywhere along the solvent capillary302, for example nearest the end 305 located away from the swab surface310, such that the solvent may be heated before it is surrounded by thesheath gas tube 312. The third heater 346 may comprise a wire heater(e.g., a tungsten wrap) and/or may be configured to heat the solventcapillary 302 and/or the solvent to at least 50° C., 100° C., 200° C.,300° C., 400° C., 500° C., 600° C., 700° C. or 800° C.

A fourth heater 348 may be arranged and adapted to heat the swab surface310, such that the sample 311 and/or the swab surface 310 may be heated.The fourth heater 348 may be located beneath a portion of the swabsurface 310 arranged and adapted to hold or contain the sample 311. Thefourth heater 348 may comprise a wire heater (e.g., a tungsten wrap)and/or may be configured to heat the sample 311 and/or swab surface 310and/or the solvent to at least 50° C., 100° C., 200° C., 300° C., 400°C., 500° C., 600° C., 700° C. or 800° C.

The swab itself may be heated so as to heat the sample 311 that islocated on the swab. For example, the fourth heater 348 may be a wireheater that is located within the swab, and may be arranged and adaptedto heat the end of the swab configured to hold and/or retain biologicsamples for analysis.

The impact of heating an ion inlet transfer capillary (such as atransfer capillary or transfer device 330 as shown in FIG. 25A) wastested on a Xevo G2-XS® quadrupole Time of Flight mass spectrometer anda Synapt G2-Si® quadrupole-ion mobility-Time of Flight massspectrometer.

The ion transfer capillary or transfer device 330 was heated using anickel wire heater in a range from 100 to 490° C. Pork liver sectionswere used and the intensities for selected fatty acids and phospholipidswere compared. Inlet capillary heating was found to have some impact onfatty acid intensities using a Xevo® mass spectrometer and no impactusing a Synapt® mass spectrometer. Intensities for the monitoredphospholipids, however, could be improved by almost two orders ofmagnitude.

FIGS. 25B-E show the impact of inlet capillary heating on absoluteintensity. FIGS. 25B and 25D relate to a Waters Synapt G2-Si® massspectrometer and FIGS. 25C and 25E relate to a Waters Xevo G2-XS® massspectrometer. Average intensities for selected fatty acids (FA),phosphatidyl ethanolamines (PE) and the most abundantphosphatidylinositol (PI) from pork liver sections are shown.

It is apparent from FIGS. 25B-E that increasing the temperature of theion transfer capillary or transfer device 330 can increase the observedintensity of phospholipids by nearly two orders of magnitude.

Ambient Ionisation Analysis of Biopsy Samples

A number of further embodiments relate to the use of ambient ionisationmass spectrometry, and in particular rapid evaporative ionisation massspectrometry (“REIMS”) and desorption electrospray ionisation (“DESI”)mass spectrometry, in the analysis of biopsy samples.

A biopsy sample is a sample of cells or tissue that is typically takenfrom a living subject and used to determine the presence or extent of adisease.

Biopsy samples can be provided using, e.g., (i) fine needle aspirationbiopsy, where a thin needle attached to a syringe is used to aspirate asmall amount of tissue, (ii) a core needle biopsy, where a hollow needleis used to withdraw cylinders (or cores) of tissue, and (iii) a surgical(or open) biopsy, where tissue is surgically cut e.g. using a scalpel. Abiopsy may optionally be incisional, excisional, or be retrieved from asurgical resection. A biopsy specimen comprises cells and may optionallybe a tissue specimen, for example, comprising or consisting of diseasedand/or non-diseased tissue.

FIG. 26 illustrates a typically core needle biopsy, in which a biopsyneedle 240 comprising a hollow needle is used to withdraw cylinders (orcores) 241 of tissue, e.g. including a tumour 242 or other area ofmedical interest.

Conventional histopathological analysis of biopsy samples involvessending the sample to a specialist lab, where the sample is prepared forexamination, and examined under a microscope. Accordingly, conventionalbiopsy analysis involves a time consuming and costly workflow.

According to various embodiments, a biopsy sample is analysed usingambient ionisation mass spectrometry, and in particular rapidevaporative ionisation mass spectrometry (“REIMS”) and/or desorptionelectrospray ionisation mass spectrometry (“DESI-MS”).

The Applicants have found that ambient ionisation mass spectrometry, andin particular rapid evaporative ionisation mass spectrometry (“REIMS”)analysis and desorption electrospray ionisation (“DESI”) massspectrometry analysis, of biopsy samples can produce pathologicallyrelevant information. Moreover, ambient ionisation mass spectrometry,and in particular rapid evaporative ionisation mass spectrometry(“REIMS”) analysis and desorption electrospray ionisation (“DESI”) massspectrometry analysis, can provide rapid, real-time, point-of-careinformation in a particularly convenient and efficient manner.

A particularly useful feature of rapid evaporative ionisation massspectrometry (“REIMS”) analysis and desorption electrospray ionisation(“DESI”) mass spectrometry analysis of biopsy samples is the relativeease of acquiring spatially resolved data using these techniques.

In particular, spatially resolved information from a biopsy sample maybe used to determine the presence, location and/or extent or size ofdiseased tissue, e.g., a tumour and/or necrotic tissue in a biopsysample.

In this regard, the Applicants have found that methods according tovarious embodiments can give more accurate data when compared with, forexample, magnetic resonance imaging (“MRI”) of tumours. In oneparticular embodiment, this may be exploited to accurately determinewhether or not the location of a tumour (e.g. close to a vein orotherwise) renders the tumour inoperable, e.g. prior to an operation. Inanother embodiment, this may be used to determine how much diseasedtissue, e.g. necrotic or cancerous tissue, needs to be, or safely canbe, removed.

Spatially resolved information from a biopsy sample may additionally oralternatively be used in determining the aggressiveness of a tumourand/or the likelihood that or degree to which a tumour will respond to aparticular treatment. For example, by analysing portions of a biopsysample adjacent to a tumour, information regarding the body's naturalresponse to the tumour can be determined, e.g. by identifying relevantbiomarkers. This information can be directly related to theaggressiveness of a tumour and/or the likelihood that or degree to whicha tumour will respond to a particular treatment.

The analysis may optionally be used to identify disease margins. Adisease margin may optionally be analysed, e.g., by analysing theconcentration of a particular cell type, e.g. a diseased, cancerous,and/or necrotic cell type, in a target region.

According to various other embodiments, information obtained from theanalysis of a biopsy sample may be used, e.g. in real-time while apatient is under anaesthesia and/or during surgery, to determine acourse of action.

Biopsy Analyser

According to an embodiment, a biopsy needle may be inserted into apatient and then the resulting biopsy sample (e.g. biopsy core) may beinserted into a dedicated channel of a mass and/or ion mobilityspectrometer.

The biopsy sample may be inserted into the channel of the mass and/orion mobility spectrometer together with the biopsy needle (i.e. that wasused to extract the sample), by itself, or together with some otherdevice for holding and/or supporting the biopsy sample.

The mass and/or ion mobility spectrometer may then analyse the sample.The mass and/or ion mobility spectrometer may, for example, performlongitudinal analysis of the biopsy needle or sample, i.e., along thelength of the biopsy needle or sample.

According to various embodiments, a one-dimensional (e.g. longitudinal)mass spectrometry image or ion image of a biopsy sample may be provided.

Rapid evaporative ionisation mass spectrometry (“REIMS”) and desorptionelectrospray ionisation mass spectrometry (“DESI-MS”) are bothparticularly suited to the analysis of biopsy samples in theseembodiments, as they can be readily used to provide spatially resolvedmass spectral data, i.e. to provide a one-dimensional (longitudinal)mass spectrometry image or ion image of a biopsy sample or core.

In various embodiments, the mass spectral analysis, e.g. including adiagnosis, may be performed in real-time and/or at the point of care(“POC”). This represents a fast and convenient method for analysingbiopsy samples.

If any issues arise from the mass analysis of the biopsy needle then asecond biopsy may immediately be performed, thereby beneficially savingthe patient from having to undergo a second biopsy procedure at a laterdate.

These embodiments are particularly relevant, for example, for liver andkidney biopsies. In these cases, the current medical practice is toinsert a catheter through the neck and to use a snare to (hopefully)capture a piece of liver or kidney. As such, various embodimentsrepresent an improved method for liver and kidney biopsies.

Biopsy Needle

According to various embodiments a biopsy needle may be provided that isarranged to collect two (or more) separate samples or portions of abiopsy sample (e.g. two (or more) biopsy cores or cylinders) at the sametime. The biopsy needle may be configured such that when it is insertedinto tissue, two (or more) separate samples or portions of the tissue(e.g. two (or more) biopsy cores or cylinders) are produced.

The biopsy needle may comprise, for example, a needle comprising a firsthollow tube or cylinder and a second hollow tube or cylinder. The firstand second hollow tubes or cylinders may be conjoined, e.g. along some,most or all of the axial length of the first and/or second hollow tubeor cylinder.

The two separate samples or portions of the tissue (e.g. the two biopsycores or cylinders) produced by the biopsy needle may comprise adjacentportions of tissue. For example, two biopsy cores or cylinders may beproduced where the first biopsy core or cylinder comprises tissue thatwas originally adjacent and/or connected to the tissue of the secondbiopsy core or cylinder, i.e. along some, most or all the axial lengthof the first and/or second biopsy core or cylinder.

According to an embodiment one of the two samples may be sent forconventional histopathological analysis and the second of the samplesmay be (e.g. substantially immediately) subjected to ambient ionisationmass spectrometry, and in particular to rapid evaporative ionisationmass spectrometry (“REIMS”) analysis and/or desorption electrosprayionisation (“DESI”) analysis.

Beneficially, ambient ionisation mass spectrometry, and in particularrapid evaporative ionisation mass spectrometry (“REIMS”) and/ordesorption electrospray ionisation (“DESI”) analysis is able to provideadditional information to the information provided by histopathology. Inparticular, rapid evaporative ionisation mass spectrometry (“REIMS”) canpotentially identify the underlying disease whereas histopathology isonly able to provide information relating to the cell chemistry.

An example application according to an embodiment is the diagnosis ofnon-alcoholic fatty liver disease (“NAFLD”).

Rapid Evaporative Ionisation Mass Spectrometry (“REIMS”) Biopsy

According to various embodiments, a biopsy needle may be provided whichcomprises a device that is configured to generate aerosol, smoke orvapour from a target. The device may comprise or form part of an ambiention or ionisation source, or the device may generate the aerosol, smokeor vapour for subsequent ionisation by an ambient ion or ionisationsource or other ionisation source.

According to one particular embodiment, a biopsy needle may be providedwhich comprises one or more electrodes, and in particular one or morerapid evaporative ionisation mass spectrometry (“REIMS”) electrodes.

According to another embodiment a biopsy needle may be provided thatcomprises a laser ionisation ion source, e.g. as describe above.According to another embodiment a biopsy needle may be provided thatcomprises an ultrasonic ablation ion source, e.g. as describe above. Thedevice or electrode(s) may be activated while the biopsy needle isinserted into a patient, e.g. to provide mass spectral data relating tothe tissue samples by the biopsy needle.

A patient undergoing a biopsy using a needle which includes an ambiention source such as a rapid evaporative ionisation mass spectrometry(“REIMS”) electrode would require an anaesthetic, but variousembodiments have a number of applications including real time diagnosisand analysis of a disease such as any of the diseases mentionedelsewhere herein, e.g., hepatitis, liver sclerosis and cancerous tissue.

According to another embodiment, the device or electrode(s) may beactivated once the biopsy needle containing a biopsy sample (core) hasbeen extracted from the patient.

According to various embodiments, the biopsy needle may comprise abiopsy needle that is arranged to collect two (or more) separate samplesor portions of a biopsy sample (e.g. two (or more) biopsy cores orcylinders) at the same time, e.g. as described above. In theseembodiments, the device or electrode(s) may be configured to (e.g.selectively) generate aerosol, smoke or vapour from one or both of thebiopsy samples. Where the device or electrode(s) is configured togenerate aerosol, smoke or vapour from only one of the biopsy samples,then the other biopsy sample may be sent for conventionalhistopathological analysis, e.g. as described above.

FIG. 27 shows an embodiment wherein a biopsy needle 250 is provided withan electrode 251 which may be at the distal end of the needle 250. Inthe embodiment illustrated in FIG. 26, the biopsy needle has beeninserted into a tumour 252 present within an internal organ 253 of apatient.

The electrode 251 is connected to an RF voltage generator (not shown).When an RF voltage is applied to the electrode 251, the electrode 251acts as an electrosurgical tool and effectively cuts the tumour 252.This causes surgical smoke or aerosol to be generated.

The surgical smoke or aerosol is aspirated into tubing 254, e.g. via oneor more fenestrations or aspiration ports (not shown), and along thelength of the tubing 254, and is passed to a vacuum chamber of a massand/or ion mobility spectrometer 255 via an atmospheric inlet 256.Aspiration of the surgical smoke or aerosol may be facilitated using aVenturi pump, e.g. driven by standard medical air or nitrogen.

The surgical smoke or aerosol may then be ionised, e.g. by impacting acollision surface which may be heated.

The resulting analyte ions may then be analysed, e.g. mass analysedand/or subjected to ion mobility analysis or separation and real timeinformation relating to the tissue or tumour 252 may be provided to auser.

The mass and/or ion mobility spectrometer 255 may include a modifiedatmospheric interface 256 which includes a collision surface which maybe positioned along and adjacent to the central axis of the largeopening of a StepWave® ion guide. As will be understood by those skilledin the art, a StepWave® ion guide comprises two conjoined ion tunnel ionguides. Each ion guide comprises a plurality of ring or other electrodeswherein ions pass through the central aperture provided by the ring orother electrodes. Transient DC voltages or potentials are applied to theelectrodes. The StepWave® ion guide is based on stacked ring ion guidetechnology and is designed to maximise ion transmission from the sourceto the mass analyser or filter. The device allows for the active removalof neutral contaminants thereby providing an enhancement to overallsignal to noise. The design enables the efficient capture of the diffuseion cloud entering a first lower stage which is then focused into anupper ion guide for transfer to the mass analyser or filter.

The collision surface which may be located within a vacuum chamber ofthe mass and/or ion mobility spectrometer 255 facilitates efficientfragmentation of molecular clusters formed in the free jet region of theatmospheric interface 256 due to the adiabatic expansion of gas enteringthe vacuum chamber and the resulting drop in temperature. Thesurface-induced dissociation of supramolecular clusters improves thesignal intensity and also alleviates the problems associated with thecontamination of ion optics.

The biopsy needle may be used in any part of the body or organs such asthe lung, liver and breast.

The biopsy needle may comprise a monopolar device and a relatively largepad acting as a return electrode may be placed underneath the patient sothat electrical current flows from the electrode 251, through thepatient, to the return electrode. Alternatively, the biopsy needle maycomprise a bipolar device, e.g. comprising two electrodes, such thatelectrical current does not flow through the patient's body. A bipolarbiopsy needle may be used, for example, where it is undesirable for anelectrical current to flow through surrounding tissue.

Although a monopolar or a bipolar electrode arrangement is particularlybeneficial, other embodiments are also contemplated wherein the biopsyneedle may comprise a multi-phase or 3-phase device and may comprise,for example, three or more separate electrodes.

A matrix may be added or mixed with the surgical smoke or aerosol priorto the surgical smoke or aerosol impacting upon the collision surface.The matrix may comprise a solvent for the surgical smoke or aerosol, andmay comprise an organic solvent and/or a volatile compound. The matrixmay comprise polar molecules, water, one or more alcohols, methanol,ethanol, isopropanol, acetone or acetonitrile. Isopropanol isparticularly beneficial to use.

The matrix which is added may additionally or alternatively comprise alockmass, lock mobility or calibration compound.

The addition of a matrix is particularly beneficial in that dissolvinganalyte in the matrix eliminates intermolecular bonding between theanalyte molecules. As such, when the dissolved analyte is collided withthe collision surface, the dissolved analyte will fragment into dropletsand any given droplet is likely to contain fewer analyte molecules thanit would if the matrix were not present. This in turn leads to a moreefficient generation of ions when the matrix in each droplet isevaporated.

According to various embodiments, the data obtained from the rapidevaporative ionisation mass spectrometry (“REIMS”) analysis of thebiopsy sample within the biopsy needle 250 may be used to ensure thatthe biopsy needle has correctly sampled a portion of tissue of interest,e.g. a portion of a tumour 252, e.g. to ensure that the needle has beeninserted to the correct depth within the patient, before the biopsyneedle 250 is removed from the patient.

Additionally or alternatively, the data obtained from the rapidevaporative ionisation mass spectrometry (“REIMS”) analysis of thebiopsy sample within the biopsy needle 250 may be used for diagnosis orcharacterisation, e.g. of the tissue or tumour 252.

The data obtained from the REI rapid evaporative ionisation massspectrometry (“REIMS”) analysis of the biopsy sample within the biopsyneedle 250 may be used on its own, or may be used to supplementsubsequent analysis (e.g. histopathological analysis) of the (e.g.remaining) biopsy sample (core).

Use of Biopsy Data to Improve Subsequent Real Time Analysis of SurgicalData Obtained Using an Ambient Ionisation Surgical Tool During aSubsequent Surgical Procedure

According to various embodiments, pre-operative characterisation ofbiopsy data may be used to improve a surgical library which may then besubsequently interrogated or used by an ambient ionisation surgicaltool, e.g. during the course of a subsequent surgical procedure.

For example, based upon the results of a biopsy analysis, subsequentmass spectral data obtained during a surgical procedure may be obtainedand/or analysed in an improved or optimal manner.

For example, if a biopsy revealed that a patient was suffering fromliver sclerosis then a subsequent surgical procedure on a portion of theliver may be performed wherein the mass spectral analysis of sampleliver tissue is optimised to distinguish between healthy liver tissueand sclerotic liver tissue. This applies mutatis mutandis to any othersuitable diseases, e.g., the other diseases mentioned elsewhere herein,particularly cancer, necrosis and the like.

It is contemplated that pre-existing surgical mass spectral (and/or ionmobility) databases, such as proprietary surgical mass spectral (and/orion mobility) databases, may be provided, and that before a surgicalprocedure is performed an appropriate surgical database may bepre-loaded, e.g. into a mass spectrometer (and/or ion mobility analyser)which is coupled to the ambient ionisation surgical tool. The databasemay then be improved or optimised using biopsy data in accordance withvarious embodiments.

Optimised Operational Parameters of an Ambient Ionisation Surgical ToolMay be Programmed or Set Dependent Upon Previously Acquired Data Such asBiopsy Data

According to various embodiments one or more operational parameters ofan ambient ionisation surgical or diagnostic tool may be arranged tovary or otherwise be optimised during a surgical or diagnosticprocedure. This may be done of the basis of previously acquired datasuch as biopsy data.

For example, according to an embodiment the energy dissipated intosurrounding tissue may be arranged to reduce as the surgical ordiagnostic device approaches a vital organ.

According to various embodiments, one or more operational parameters ofan ambient ionisation surgical tool may be set based upon previouslyacquired data such as biopsy data.

For example, one or more operational parameters of an ambient ionisationsurgical tool may be set based upon the type or grade of canceroustissue identified during a biopsy or based upon the nature of thediseased tissue identified during a biopsy.

In these embodiments, the cancerous biological tissue or the tumour maycomprise, for example: (i) grade I, grade II, grade III or grade IVcancerous tissue; (ii) metastatic cancerous tissue; (iii) mixed gradecancerous tissue; or (iv) a sub-grade cancerous tissue.

Different operational parameters may be used depending upon the type oftissue being operated on, such as depending on whether healthy tissue,clearly diseased (e.g. cancerous) tissue or tissue at the disease (e.g.cancer) margin is being operated on.

According to various embodiments the biopsy data may include spatialinformation and hence the variation of tissue as a function of depthwithin an organ may be determined. Accordingly, previously acquiredbiopsy data may be used to set various operational parameters of anambient ionisation surgical tool, e.g., as the surgical tool movesdeeper into an organ.

Furthermore, various ionisation parameters may be varied, e.g. as theambient ionisation surgical tool moves deeper into an organ.

For example, as an ambient ionisation surgical tool makes an initial cutinto an organ, one or more ionisation parameters (e.g., composition ofmatrix added to aerosol, smoke or vapour released from the tissue,temperature of an ionisation collision surface, voltage applied to anionisation collision surface, etc.) may be optimised for the surgicalconditions (e.g., initial blood loss, tissue composition, etc.)experienced when initially cutting into the organ. As the ambientionisation surgical tool moves deeper into the organ the optimumionisation parameters for the surgical tool may change reflecting, e.g.,a different degree of blood and a different composition of the tissue.Accordingly, one or more ionisation parameters (e.g., composition ofmatrix added to aerosol, smoke or vapour released from the tissue,temperature of an ionisation collision surface, voltage applied to anionisation collision surface, etc.) may be arranged to also change tomatch the changing surgical conditions.

Numerous different embodiments are contemplated wherein variousoperational parameters of a surgical device such as an ambientionisation ion source (e.g. a rapid evaporative ionisation massspectrometry (“REIMS”) ion source) may be varied on the basis ofpreviously acquired data such as biopsy data.

Analysing Sample Spectra

A list of analysis techniques which may be used in accordance withvarious embodiments is given in the following table:

Analysis Techniques Univariate Analysis Multivariate Analysis PrincipalComponent Analysis (PCA) Linear Discriminant Analysis (LDA) MaximumMargin Criteria (MMC) Library Based Analysis Soft Independent ModellingOf Class Analogy (SIMCA) Factor Analysis (FA) Recursive Partitioning(Decision Trees) Random Forests Independent Component Analysis (ICA)Partial Least Squares Discriminant Analysis (PLS-DA) Orthogonal (PartialLeast Squares) Projections To Latent Structures (OPLS) OPLS DiscriminantAnalysis (OPLS-DA) Support Vector Machines (SVM) (Artificial) NeuralNetworks Multilayer Perceptron Radial Basis Function (RBF) NetworksBayesian Analysis Cluster Analysis Kernelized Methods SubspaceDiscriminant Analysis K-Nearest Neighbours (KNN) Quadratic DiscriminantAnalysis (QDA) Probabilistic Principal Component Analysis (PPCA) Nonnegative matrix factorisation K-means factorisation Fuzzy c-meansfactorisation Discriminant Analysis (DA)

Combinations of the foregoing analysis approaches can also be used, suchas PCA-LDA, PCA-MMC, PLS-LDA, etc.

Analysing the sample spectra can comprise unsupervised analysis fordimensionality reduction followed by supervised analysis forclassification.

By way of example, a number of different analysis techniques will now bedescribed in more detail.

Multivariate Analysis—Developing a Model for Classification

By way of example, a method of building a classification model usingmultivariate analysis of plural reference sample spectra will now bedescribed.

FIG. 28 shows a method 1500 of building a classification model usingmultivariate analysis. In this example, the method comprises a step 1502of obtaining plural sets of intensity values for reference samplespectra. The method then comprises a step 1504 of unsupervised principalcomponent analysis (PCA) followed by a step 1506 of supervised lineardiscriminant analysis (LDA). This approach may be referred to herein asPCA-LDA. Other multivariate analysis approaches may be used, such asPCA-MMC. The PCA-LDA model is then output, for example to storage, instep 1508.

The multivariate analysis such as this can provide a classificationmodel that allows a sample (such as an aerosol, smoke or vapour sample,a biological sample, etc.) to be classified using one or more samplespectra obtained from the sample. The multivariate analysis will now bedescribed in more detail with reference to a simple example.

FIG. 29 shows a set of reference sample spectra obtained from twoclasses of known reference samples. The classes may be any one or moreof the classes of target described herein. However, for simplicity, inthis example the two classes will be referred as a left-hand class and aright-hand class.

Each of the reference sample spectra has been pre-processed in order toderive a set of three reference peak-intensity values for respectivemass to charge ratios in that reference sample spectrum. Although onlythree reference peak-intensity values are shown, it will be appreciatedthat many more reference peak-intensity values (e.g., ˜100 referencepeak-intensity values) may be derived for a corresponding number of massto charge ratios in each of the reference sample spectra. In otherembodiments, the reference peak-intensity values may correspond to:masses; mass to charge ratios; ion mobilities (drift times); and/oroperational parameters.

FIG. 30 shows a multivariate space having three dimensions defined byintensity axes. Each of the dimensions or intensity axes corresponds tothe peak-intensity at a particular mass to charge ratio. Again, it willbe appreciated that there may be many more dimensions or intensity axes(e.g., ˜100 dimensions or intensity axes) in the multivariate space. Themultivariate space comprises plural reference points, with eachreference point corresponding to a reference sample spectrum, i.e., thepeak-intensity values of each reference sample spectrum provide theco-ordinates for the reference points in the multivariate space.

The set of reference sample spectra may be represented by a referencematrix D having rows associated with respective reference samplespectra, columns associated with respective mass to charge ratios, andthe elements of the matrix being the peak-intensity values for therespective mass to charge ratios of the respective reference samplespectra.

In many cases, the large number of dimensions in the multivariate spaceand matrix D can make it difficult to group the reference sample spectrainto classes. PCA may accordingly be carried out on the matrix D inorder to calculate a PCA model that defines a PCA space having a reducednumber of one or more dimensions defined by principal component axes.The principal components may be selected to be those that comprise or“explain” the largest variance in the matrix D and that cumulativelyexplain a threshold amount of the variance in the matrix D.

FIG. 31 shows how the cumulative variance may increase as a function ofthe number n of principal components in the PCA model. The thresholdamount of the variance may be selected as desired.

The PCA model may be calculated from the matrix D using a non-lineariterative partial least squares (NIPALS) algorithm or singular valuedecomposition, the details of which are known to the skilled person andso will not be described herein in detail. Other methods of calculatingthe PCA model may be used.

The resultant PCA model may be defined by a PCA scores matrix S and aPCA loadings matrix L. The PCA may also produce an error matrix E, whichcontains the variance not explained by the PCA model. The relationshipbetween D, S, L and E may be:

D=SL ^(T) +E   (1)

FIG. 32 shows the resultant PCA space for the reference sample spectraof FIGS. 29 and 30. In this example, the PCA model has two principalcomponents PCo and PCi and the PCA space therefore has two dimensionsdefined by two principal component axes. However, a lesser or greaternumber of principal components may be included in the PCA model asdesired. It is generally desired that the number of principal componentsis at least one less than the number of dimensions in the multivariatespace.

The PCA space comprises plural transformed reference points or PCAscores, with each transformed reference point or PCA score correspondingto a reference sample spectrum of FIG. 29 and therefore to a referencepoint of FIG. 30.

As is shown in FIG. 32, the reduced dimensionality of the PCA spacemakes it easier to group the reference sample spectra into the twoclasses. Any outliers may also be identified and removed from theclassification model at this stage.

Further supervised multivariate analysis, such as multi-class LDA ormaximum margin criteria (MMC), in the PCA space may then be performed soas to define classes and, optionally, further reduce the dimensionality.

As will be appreciated by the skilled person, multi-class LDA seeks tomaximise the ratio of the variance between classes to the variancewithin classes (i.e., so as to give the largest possible distancebetween the most compact classes possible). The details of LDA are knownto the skilled person and so will not be described herein in detail.

The resultant PCA-LDA model may be defined by a transformation matrix U,which may be derived from the PCA scores matrix S and class assignmentsfor each of the transformed spectra contained therein by solving ageneralised eigenvalue problem.

The transformation of the scores S from the original PCA space into thenew LDA space may then be given by:

Z=SU   (2)

where the matrix Z contains the scores transformed into the LDA space.

FIG. 33 shows a PCA-LDA space having a single dimension or axis, whereinthe LDA is performed in the PCA space of FIG. 32. As is shown in FIG.33, the LDA space comprises plural further transformed reference pointsor PCA-LDA scores, with each further transformed reference pointcorresponding to a transformed reference point or PCA score of FIG. 32.

In this example, the further reduced dimensionality of the PCA-LDA spacemakes it even easier to group the reference sample spectra into the twoclasses. Each class in the PCA-LDA model may be defined by itstransformed class average and covariance matrix or one or morehyperplanes (including points, lines, planes or higher orderhyperplanes) or hypersurfaces or Voronoi cells in the PCA-LDA space.

The PCA loadings matrix L, the LDA matrix U and transformed classaverages and covariance matrices or hyperplanes or hypersurfaces orVoronoi cells may be output to a database for later use in classifyingan aerosol, smoke or vapour sample.

The transformed covariance matrix in the LDA space V′_(g) for class gmay be given by

V′_(g)=U^(T)V_(g)U   (3)

where V_(g) are the class covariance matrices in the PCA space.

The transformed class average position z_(g) for class g may be given by

s_(g)U=z_(g)   (4)

where s_(g) is the class average position in the PCA space.

Multivariate Analysis—Using a Model for Classification

By way of example, a method of using a classification model to classifya sample (such as an aerosol, smoke or vapour sample) will now bedescribed.

FIG. 34 shows a method 2100 of using a classification model. In thisexample, the method comprises a step 2102 of obtaining a set ofintensity values for a sample spectrum. The method then comprises a step2104 of projecting the set of intensity values for the sample spectruminto PCA-LDA model space. Other classification model spaces may be used,such as PCA-MMC. The sample spectrum is then classified at step 2106based on the project position and the classification is then output instep 2108.

Classification of a sample (e.g. an aerosol, smoke or vapour sample)will now be described in more detail with reference to the simplePCA-LDA model described above.

FIG. 35 shows a sample spectrum obtained from an unknown aerosol, smokeor vapour sample. The sample spectrum has been pre-processed in order toderive a set of three sample peak-intensity values for respective massto charge ratios. As mentioned above, although only three samplepeak-intensity values are shown, it will be appreciated that many moresample peak-intensity values (e.g., ˜100 sample peak-intensity values)may be derived at many more corresponding mass to charge ratios for thesample spectrum. Also, as mentioned above, in other embodiments, thesample peak-intensity values may correspond to: masses; mass to chargeratios; ion mobilities (drift times); and/or operational parameters.

The sample spectrum may be represented by a sample vector dx, with theelements of the vector being the peak-intensity values for therespective mass to charge ratios. A transformed PCA vector sx for thesample spectrum can be obtained as follows:

dxL=sx   (5)

Then, a transformed PCA-LDA vector zx for the sample spectrum can beobtained as follows:

s_(X)U=z_(X)   (6)

FIG. 36 again shows the PCA-LDA space of FIG. 33. However, the PCA-LDAspace of FIG. 36 further comprises the projected sample point,corresponding to the transformed PCA-LDA vector z_(X), derived from thepeak intensity values of the sample spectrum of FIG. 35.

In this example, the projected sample point is to one side of ahyperplane between the classes that relates to the right-hand class, andso the sample (aerosol, smoke or vapour sample) may be classified asbelonging to the right-hand class.

Alternatively, the Mahalanobis distance from the class centres in theLDA space may be used, where the Mahalanobis distance of the point z_(x)from the centre of class g may be given by the square root of:

(z _(x) −z _(g))^(T)(V′ _(g))⁻¹(z _(x) −z _(g))   (8)

and the data vector d_(x) may be assigned to the class for which thisdistance is smallest.

In addition, treating each class as a multivariate Gaussian, aprobability of membership of the data vector to each class may becalculated.

Library Based Analysis—Developing a Library for Classification

By way of example, a method of building a classification library usingplural input reference sample spectra will now be described.

FIG. 37 shows a method 2400 of building a classification library. Inthis example, the method comprises a step 2402 of obtaining plural inputreference sample spectra and a step 2404 of deriving metadata from theplural input reference sample spectra for each class of sample. Themethod then comprises a step 2404 of storing the metadata for each classof sample as a separate library entry. The classification library isthen output, for example to electronic storage, in step 2406.

A classification library such as this allows a sample (e.g. an aerosol,smoke or vapour sample) to be classified using one or more samplespectra obtained from the sample. The library based analysis will now bedescribed in more detail with reference to an example.

In this example, each entry in the classification library is createdfrom plural pre-processed reference sample spectra that arerepresentative of a class. In this example, the reference sample spectrafor a class are pre-processed according to the following procedure:

First, a re-binning process is performed. In this embodiment, the dataare resampled onto a logarithmic grid with abscissae:

$\begin{matrix}{x_{i} = \left\lfloor {N_{chan}\log {\frac{m}{M_{\min}}/\log}\frac{M_{\max}}{M_{\min}}} \right\rfloor} & (7)\end{matrix}$

where N_(chan) is a selected value and └x┘ denotes the nearest integerbelow x. In one example, N_(chan) is 2¹² or 4096.

Then, a background subtraction process is performed. In this embodiment,a cubic spline with k knots is then constructed such that p % of thedata between each pair of knots lies below the curve. This curve is thensubtracted from the data. In one example, k is 32. In one example, p is5.

A constant value corresponding to the q % quantile of the intensitysubtracted data is then subtracted from each intensity. Positive andnegative values are retained. In one example, q is 45.

Then, a normalisation process is performed. In this embodiment, the dataare normalised to have mean y _(i). In one example, y _(i)=1.

An entry in the library then consists of metadata in the form of amedian spectrum value μ_(i) and a deviation value D_(i) for each of theN_(chan) points in the spectrum.

The likelihood for the i'th channel is given by:

$\begin{matrix}{{\Pr \left( {{y_{i}\mu_{i}},D_{i}} \right)} = {\frac{1}{D_{i}}\frac{C^{C - {1/2}}{\Gamma (C)}}{\sqrt{\pi}{\Gamma \left( {C - {1/2}} \right)}}\frac{1}{\left( {C + \frac{\left( {y_{i} - \mu_{i}} \right)^{2}}{D_{i}^{2}}} \right)^{C}}}} & (8)\end{matrix}$

where ½<C<∞ and where Γ(C) is the gamma function.

The above equation is a generalised Cauchy distribution which reduces toa standard Cauchy distribution for C=1 and becomes a Gaussian (normal)distribution as C→∞. The parameter D_(i) controls the width of thedistribution (in the Gaussian limit D_(i)=σ_(i) is simply the standarddeviation) while the global value C controls the size of the tails.

In one example, C is 3/2, which lies between Cauchy and Gaussian, sothat the likelihood becomes:

$\begin{matrix}{{\Pr \left( {{y_{i}\mu_{i}},D_{i}} \right)} = {\frac{3}{4}\frac{1}{D_{i}}\frac{1}{\left( {{3/2} + {\left( {y_{i} - \mu_{i}} \right)^{2}/D_{i}^{2}}} \right)^{3/2}}}} & (9)\end{matrix}$

For each library entry, the parameters μ_(i) are set to the median ofthe list of values in the i'th channel of the input reference samplespectra while the deviation D_(i) is taken to be the interquartile rangeof these values divided by √2. This choice can ensure that thelikelihood for the i'th channel has the same interquartile range as theinput data, with the use of quantiles providing some protection againstoutlying data.

Library Based Analysis—Using a Library for Classification

By way of example, a method of using a classification library toclassify a sample (e.g. an aerosol, smoke or vapour sample) will now bedescribed.

FIG. 38 shows a method 2500 of using a classification library. In thisexample, the method comprises a step 2502 of obtaining a set of pluralsample spectra. The method then comprises a step 2504 of calculating aprobability or classification score for the set of plural sample spectrafor each class of sample using metadata for the class entry in theclassification library. The sample spectra are then classified at step2506 and the classification is then output in step 2508.

Classification of a sample (e.g. an aerosol, smoke or vapour sample)will now be described in more detail with reference to theclassification library described above.

In this example, an unknown sample spectrum y is the median spectrum ofa set of plural sample spectra. Taking the median spectrum y can protectagainst outlying data on a channel by channel basis.

The likelihood L_(s) for the input data given the library entry s isthen given by:

L _(s) =Pr(γ|μ,D)=Π_(i=1) ^(N) ^(chan) Pr(γ_(i)/μ_(i) , D _(i))   (10)

where μ_(i) and D_(i) are, respectively, the library median values anddeviation values for channel i. The likelihoods L_(s) may be calculatedas log likelihoods for numerical safety.

The likelihoods L_(s) are then normalised over all candidate classes ‘s’to give probabilities, assuming a uniform prior probability over theclasses. The resulting probability for the class {tilde over (s)} isgiven by:

$\begin{matrix}{{\Pr \left( {\overset{\sim}{s}y} \right)} = \frac{L_{\overset{\sim}{S}}^{({1/F})}}{\sum_{S}L_{S}^{({1/F})}}} & (11)\end{matrix}$

The exponent (1/F) can soften the probabilities which may otherwise betoo definitive. In one example, F=100. These probabilities may beexpressed as percentages, e.g., in a user interface. Alternatively, RMSclassification scores R_(s) may be calculated using the same mediansample values and derivation values from the library:

$\begin{matrix}{{R_{S}\left( {y,\mu,D} \right)} = \sqrt{\frac{1}{N_{chan}}{\sum_{i = 1}^{N_{chan}}\frac{\left( {y_{i} - \mu_{i}} \right)^{2}}{D_{i}^{2}}}}} & (12)\end{matrix}$

Again, the scores R_(s) are normalised over all candidate classes ‘s’.

The sample (e.g. aerosol, smoke or vapour sample) may then be classifiedas belonging to the class having the highest probability and/or highestRMS classification score.

Methods of Analysis, E.g., Methods of Medical Treatment, Surgery andDiagnosis and Non-Medical Methods

Various different embodiments are contemplated. According to someembodiments the methods disclosed above may be performed on in vivo, exvivo or in vitro tissue. The tissue may comprise human or non-humananimal tissue. Embodiments are contemplated wherein the target maycomprise biological tissue, a bacterial or fungal colony or moregenerally an organic target such as a plastic).

Various embodiments are contemplated wherein analyte ions generated byan ambient ionisation ion source are then subjected either to: (i) massanalysis by a mass analyser or filter such as a quadrupole mass analyseror a Time of Flight mass analyser; (ii) ion mobility analysis (IMS)and/or differential ion mobility analysis (DMA) and/or Field AsymmetricIon Mobility Spectrometry (FAIMS) analysis; and/or (iii) a combinationof firstly (or vice versa) ion mobility analysis (IMS) and/ordifferential ion mobility analysis (DMA) and/or Field Asymmetric IonMobility Spectrometry (FAIMS) analysis followed by secondly (or viceversa) mass analysis by a mass analyser or filter such as a quadrupolemass analyser or a Time of Flight mass analyser. Various embodimentsalso relate to an ion mobility spectrometer and/or mass analyser and amethod of ion mobility spectrometry and/or method of mass analysis. Ionmobility analysis may be performed prior to mass to charge ratioanalysis or vice versa.

Various references are made in the present application to mass analysis,mass analysers, mass analysing, mass spectrometric data, massspectrometers and other related terms referring to apparatus and methodsfor determining the mass or mass to charge of analyte ions. It should beunderstood that it is equally contemplated that the present inventionmay extend to ion mobility analysis, ion mobility analysers, ionmobility analysing, ion mobility data, ion mobility spectrometers, ionmobility separators and other related terms referring to apparatus andmethods for determining the ion mobility, differential ion mobility,collision cross section or interaction cross section of analyte ions.Furthermore, it should also be understood that embodiments arecontemplated wherein analyte ions may be subjected to a combination ofboth ion mobility analysis and mass analysis i.e. that both (a) the ionmobility, differential ion mobility, collision cross section orinteraction cross section of analyte ions together with (b) the mass tocharge of analyte ions is determined. Accordingly, hybrid ionmobility-mass spectrometry (IMS-MS) and mass spectrometry-ion mobility(MS-IMS) embodiments are contemplated wherein both the ion mobility andmass to charge ratio of analyte ions generated e.g. by an ambientionisation ion source are determined. Ion mobility analysis may beperformed prior to mass to charge ratio analysis or vice versa.Furthermore, it should be understood that embodiments are contemplatedwherein references to mass spectrometric data and databases comprisingmass spectrometric data should also be understood as encompassing ionmobility data and differential ion mobility data etc. and databasescomprising ion mobility data and differential ion mobility data etc.(either in isolation or in combination with mass spectrometric data).

Various surgical, therapeutic, medical treatment and diagnostic methodsare contemplated.

However, other embodiments are contemplated which relate to non-surgicaland non-therapeutic methods of mass spectrometry which are not performedon in vivo tissue. Other related embodiments are contemplated which areperformed in an extracorporeal manner such that they are performedoutside of the human or animal body.

Further embodiments are contemplated wherein the methods are performedon a non-living human or animal, for example, as part of an autopsyprocedure.

According to some embodiments the methods disclosed above may be carriedout on a “target”, which may optionally be a subject or a specimenderived from a subject, e.g. the biological material on a swab or abiopsy specimen.

According to various embodiments the target may comprise biologicalmatter or organic matter (including a plastic). According to variousembodiments the target may comprise one or more bacterial coloniesand/or one or more fungal colonies.

The “subject” may be a human or a non-human animal. The subject may bealive or dead. If the method is carried out on a living subject, then itmay be referred to as an in vivo method. If the method is carried out ona specimen, then it may be referred to as an in vitro or ex vivo method.

Optionally, the non-human animal may be a mammal, optionally selected,for example, from any livestock, domestic or laboratory animal, such as,mice, guinea pigs, hamsters, rats, goats, pigs, cats, dogs, sheep,rabbits, cows, horses and/or monkeys. Optionally, it may be an insect,bird, or fish, e.g. a fly, or a worm.

The method may optionally be carried out on an in vivo target, i.e. on aliving subject. For example, it may be carried out by using a thermalablation method.

Alternatively or in addition, it may optionally be carried out on a deadsubject, for example as part of an autopsy or a necropathy.

Alternatively or in addition, it may optionally be carried out on an exvivo or in vitro target, e.g., on a specimen. The specimen mayoptionally be a provided specimen, i.e. a specimen that was previouslyobtained or removed from a subject. Optionally, the method may include astep of obtaining a specimen from a subject. The term “tissue” is usedherein interchangeably with “biological tissue” and is used herein todenote a structure of cells, which may optionally be, for example, astructure, an organ, or part of a structure of organ. The tissue may bein vivo, ex vivo or in vitro.

Examples of tissues that may optionally be analysed are adrenal glandtissue, appendix tissue, bladder tissue, bone, bowel tissue, braintissue, breast tissue, bronchi, ear tissue, oesophagus tissue, eyetissue, endometrioid tissue, gall bladder tissue, genital tissue, hearttissue, hypothalamus tissue, kidney tissue, large intestine tissue,intestinal tissue, larynx tissue, liver tissue, lung tissue, lymphnodes, mouth tissue, nose tissue, pancreatic tissue, parathyroid glandtissue, pituitary gland tissue, prostate tissue, rectal tissue, salivarygland tissue, skeletal muscle tissue, skin tissue, small intestinetissue, spinal cord, spleen tissue, stomach tissue, thymus gland tissue,trachea tissue, thyroid tissue, ureter tissue, urethra tissue, soft andconnective tissue, peritoneal tissue, blood vessel tissue and/or fattissue; (ii) grade I, grade II, grade III or grade IV cancerous tissue;(iii) metastatic cancerous tissue; (iv) mixed grade cancerous tissue;(v) a sub-grade cancerous tissue; (vi) healthy or normal tissue; or(vii) cancerous or abnormal tissue.

The analysis may optionally relate to a disease or condition, such as,any of the diseases or conditions listed in this section and/orelsewhere herein. The terms “disease” and “condition” are usedinterchangeably herein.

The disease may be a skin condition, which may optionally be selected,for example, from Acne, Alopecia, Boils, Bowen's Disease, Bullouspemphigoid (BP), Carbuncle, Cellulitis, Chilblains, Cysts, Darier'sdisease, Dermatitis, Dermatomyositis, Eczema, Erythema, Exanthema,Folliculitis, Frostbite, Herpes, Ichthyosis, Impetigo, Intertrigo,Keratosis, Lichen planus, Linear IgA disease, Melanoma, Moles,Onychomycosis, Papillioma, Petechiae, Prurigo, Psoriasis, Rosacea,Scabies, Scleroderma, Sebaceous Cyst, Shingles/ Chickenpox,Telangiectasia, Urticaria (Hives), Warts and/or Xeroderma.

The disease may be a liver condition, which may optionally be selectedfrom, for example, hepatitis, fatty liver disease, alcoholic hepatitis,liver sclerosis and/or cirrhosis. Lung conditions may optionally beselected from, for example, Asthma, Atelectasis, Bronchitis, Chronicobstructive pulmonary disease (COPD), Emphysema, Lung cancer, Pneumonia,Pulmonary edema, Pneumothorax, and/or Pulmonary embolus.

The thyroid gland is an endocrine gland which normally producesthyroxine (T4) and triiodothyronine (T3). The disease may be a thyroidcondition, which may optionally be, e.g., hypothyroidism orhyperthyroidism.

The disease may be a cancer or tumour; these terms are usedinterchangeably herein, The cancer or tumour may optionally be selectedfrom, for example, carcinomas, sarcomas, leukaemias, lymphomas andgliomas.

More particularly, it may optionally be selected from, for example,Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML),Adrenocortical Carcinoma, adenoma, Anal Cancer, Appendix Cancer,Astrocytomas, Basal Cell Carcinoma, Bile Duct Cancer, Birch-Hirschfield,Blastoma, Bladder Cancer, Bone Cancer, Ewing Sarcoma, Osteosarcoma,Malignant Fibrous Histiocytoma, Brain Stem Glioma, Brain cancer,glioblastoma multiforme (“GBM”), Astrocytomas, Spinal Cord cancer,Craniopharyngioma, Breast Cancer, Bronchial Tumour, Burkitt Lymphoma,Carcinoid Tumour, Cervical Cancer, Cholangiocarcinoma, Chordoma, ChronicLymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), ChronicMyeloproliferative Neoplasms, Colon Cancer, Colorectal Cancer,Craniopharyngioma, Childhood, Ductal Carcinoma In Situ (DCIS),Endometrial Cancer, Ependymoma, Esophageal Cancer,Esthesioneuroblastoma, Fibroadenoma, Intraocular Melanoma,Retinoblastoma, Fallopian Tube Cancer, Gallbladder Cancer, Gastric(Stomach) Cancer, Germinoma, Hairy Cell Leukemia, Head and Neck Cancer,Heart Cancer, Heptacarcinoma, Hodgkin Lymphoma, Hypopharyngeal Cancer,Kahler, Kaposi Sarcoma, Kidney cancer, Laryngeal Cancer, Leiomyoma, Lipand Oral Cavity Cancer, Liver Cancer, Lung Cancer (such as, Non-SmallCell or Small Cell), Lymphoma, Lymphoblastoma, Male Breast Cancer,Malignant Fibrous Histiocytoma of Bone, Melanoma, Melanocarcinoma,Medulloblastoma, Merkel Cell Carcinoma, Mesothelioma, Mouth Cancer,Myeloma, Multiple Myeloma, Mycosis Fungoides, Myeloproliferativedisorder, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Nephroblastoma, Non-Hodgkin Lymphoma, OralCancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, PancreaticCancer, Papillomatosis, Paraganglioma, Parathyroid Cancer, PenileCancer, Peritoneal cancer, Pharyngeal Cancer, Pheochromocytoma,Pineoblastoma, Pituitary Tumour, Prostate Cancer, Rectal Cancer,Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, SézarySyndrome, Skin Cancer, Seminoma, Teratoma, Testicular Cancer, ThroatCancer, Thyroid Cancer, thoracic cancer, Urethral Cancer, VaginalCancer, Vulvar Cancer, Waldenstrom macroglobulinemia, and/or Wilm'stumour. In the above list, any reference to a “cancer” or a “tumour”should be understood to include a reference to a “cancer and/or atumour” of that type.

Optionally, the brain cancer may be glioblastoma multiforme,glioblastoma, giant cell glioblastoma, recurrent gliobastoma, anaplasticastrocytoma, oligodendroglioma and/or diffuse astrocytoma.

If the cancer is breast cancer, it may optionally be selected from, forexample, ductal carcinoma in situ (DCIS), lobular carcinoma in situ(LCIS), Invasive breast cancer (NST), Invasive lobular breast cancer,Inflammatory breast cancer, breast cancer associated with Paget'sdisease and angiosarcoma of the breast.

The cancer may be caused by, associated with, and/or characterised by amutation or other genetic variation, which may optionally result in thealtered expression of a molecule, e.g. a molecule comprising orconsisting of a lipid, such as, a glycolipid or phospholipid; acarbohydrate; DNA; RNA; a protein; a polypeptide, such as, a ribosomalpeptide or a non-ribosomal peptide; an oligopeptide; a lipoprotein; alipopeptide; an amino acid; and/or a chemical compound, optionally anorganic chemical compound. More particularly, a mutation may optionallyresult in the altered expression of a protein and/or metabolite.

A cancer may optionally express one or more metabolites that may serveas a biomarker for that cancer. For example, optionally a metabolitesuch as succinate, fumarate, 2-HG, and/or any of the other metabolitesmentioned herein may accumulate in a cancer.

Subtypes of cancer may optionally be identified, e.g., based on suchaltered expression. For example, a cancer may optionally be identifiedas being of a particular subtype based on the expression, or lackthereof, of a receptor, e.g., selected from estrogen receptors (ER),progesterone receptors (PR) and human epidermal growth factor receptor 2(HER2). A cancer may therefore, for example, be referred to as ERnegative if it lacks expression of ER; or be referred to astriple-negative breast cancer (TNBC), if it is ER-, PR- and Her2-.

The mutation may optionally, e.g., be in a gene encoding isocitratedehydrogenase 1 (IDH1) and/or 2 (IDH2) yielding mutant enzymes capableof converting alpha-ketoglutarate to 2- hydroxyglutarate (2-HG). Such amutation may optionally be present, e.g., in a glioma, intrahepaticcholangiocarcinoma, acute myelogenous leukaemia (AML) and/orchondrosarcomas. 2-HG may thus be referred to as an oncometabolite. 2-HGmay be present in very small small amounts in normal tissues, whereas itmay be present in high concentrations, e.g., several micromoles per gramof tumor, in mutant tumours.

Optionally, the type, subtype, malignancy, stage, grade, genotype and/orphenotype of a cancer may be analysed via the methods disclosed herein.

Optionally, a lesion, optionally of any of the tissues mentioned herein,may be analysed. A lesion is region in a tissue which is abnormal as aconsequence of, e.g., injury or disease. The lesion may, for example, beselected from a wound, an ulcer, an abscess, and/or a tumour. The lesionmay, for example, be a diabetic lesion, such as, a diabetic limb ordigit, or a diabetic ulcer.

Further examples of tissues that may be analysed are discussed elsewhereherein, e.g., tissue affected by, or in the vicinity of, cancer,necrosis, microbes and the like. For example, the tissue may optionallycomprise or consist of mucosa, which is discussed elsewhere herein.

The method may optionally involve the analysis of necrosis, e.g. theanalysis of tissue to determine whether a particular tissue is necroticor healthy. Thus, the margin between healthy and necrotic tissue mayoptionally be analysed. This analysis may be used to assist in decidingwhich tissue to remove surgically and which tissue may be viable enoughto be retained by the subject.

“Necrosis” is unprogrammed cell death, which may be contrasted withapoptosis, which is a form of programmed cell death.

Necrosis typically involves damage to the cell membrane and/or damage tointracellular compartments, such as, lysosomes. Necrosis is typicallyaccompanied by the release of intracellular molecules, such as, enzymes,organic chemical molecules and the like. For example, it may include therelease of the lysosomal enzymes. The release of such molecules maycause inflammation and/or damage to neighbouring cells.

The necrosis may optionally be caused by, or associated with, forexample, injury, infection, cancer, infarction, toxins, inflammation,lack of proper care to a wound site, frostbite, diabetes, and/orarteriosclerosis. Optionally, the necrosis may be necrosis of cancerousor non- cancerous tissue.

The necrosis may optionally, for example, be coagulative, liquefactive,caseous, fat necrosis, fibrinoid necrosis and/or gangrenous necrosis.

Optionally, the method may involve the analysis of the cellularcomposition of a tissue. For example, the proportion of one or moreparticular cell types may be analysed. The cell types may optionally beselected from any known cell types, e.g., any of the cell typesmentioned herein.

As mentioned above, the subject or biological material on which themethods disclosed herein may be performed may be referred to as a“target”. A target may comprise one or more “target entities”. The term“target entity” is used herein to refer to the entity which it isdesired to analyse within the target. Thus, any reference to a “target”should be understood to mean a target comprising one or more differenttarget entities. Thus, the target entity may, e.g., be a cell, microbeand/or compound. For example, the target may be a mucosal specimen,faecal specimen or body fluid on a swab and the target entity may becancer cells and/or microbes within that mucosal specimen, faecalspecimen or body fluid.

The terms “analysis”, “analysing” and derivatives of these terms areused herein to encompass any of the following: detection of a targetentity; identification of a target entity; characterisation of a targetentity; determination of the location of target entity; determination ofa status, e.g. a disease status; and/or determination of a marginbetween two different disease or tissue types and the like.

The analysis may be qualitative and/or quantitative. Thus, optionally,the analysis may involve determining the concentration, percentage,relative abundance or the like of the target entity. For example, thepercentage of cancer cells within a tissue, the relative abundance ofmicrobes in a target, and/or the concentration of a compound may beanalysed. Optionally, an increase or decrease in a target entity may beanalysed.

The terms “detection”, “detecting” and derivations of these terms areused interchangeably herein to mean that the presence or absence of atarget entity or biomarker therefor is determined.

The terms “identify”, “identification” and derivations of these termsare used interchangeably herein to mean that information about theidentity of a target entity or biomarker therefor is obtained. This mayoptionally be the determination of the identity, and/or the confirmationof the identity. This may optionally include information about theprecise identity of the target entity or biomarker therefor. However, itmay alternatively include information that allows the target entity tobe identified as falling into a particular classification, as discussedelsewhere herein.

By “identifying” a microbe is meant that at least some information aboutthe identity is obtained, which may, for example, be at any taxonomiclevel.

By “identifying” a cell is meant that at least some information aboutthe cell type is obtained. By “identifying” a diseased cell is meantthat it is determined or confirmed that a cell is diseased.

By “identifying” a compound is meant that at least some informationabout the structure and/or function of the compound is obtained, e.g.,the information may optionally allow a compound to be identified ascomprising or consisting of a compound selected from any of the typesdisclosed herein, and/or as being characterised by one or more of thefunctional groups disclosed herein.

The terms “diagnosis” or “diagnosing” and derivations of these terms asused herein refer to the determination whether or not a subject issuffering from a disease. Optionally, the method may involve analysing atarget and, on the basis of one or more of the following making adiagnosis that a subject is or is not suffering from a particulardisease: detecting a target entity; identifying a target entity;detecting an increase in a target entity; detecting a decrease in atarget entity.

An increase or decrease may be determined by reference to a suitablereference, comparator or control. For example, it is known how manyinflammatory cells or inflammatory molecules are typically present inthe tissue of a healthy individual, so an increase in inflammatory cellsor inflammatory molecules in a target may easily be determined bycomparing it to a healthy control.

The term “monitoring” and derivations of this term as used herein referto the determination whether any changes take place/have taken place.Typically, it is determined whether any changes have taken place overtime, i.e. since a previous time point. The change may, for example, bethe development and/or progression of a disease, such as, any of thediseases mentioned. Optionally, the method may involve analysing atarget and, on the basis of one or more of the following monitoring asubject or disease: detecting a target entity; identifying a targetentity; detecting an increase in a target entity; detecting a decreasein a target entity.

The term “prognosis” and derivations of this term as used herein referto risk prediction of the severity of disease or of the probable courseand clinical outcome associated with a disease. Thus, the term “methodof prognosis” as used herein refers to methods by which the skilledperson can estimate and/or determine a probability that a given outcomewill occur. The outcome to which the prognosis relates may be morbidityand/or mortality. In particular, the prognosis may relate to“progression-free survival” (PFS), which is the length of time that asubject lives with the disease without the disease progressing. Thus,PFS may, for example, be the time from the start of therapy to the dateof disease progression, or the time from the end of therapy to the dateof disease progression. Optionally, the prognosis may relate to “overallsurvival” which is the length of time that the subject is expected tolive until death.

Optionally, the method may involve analysing a target and, on the basisof one or more of the following making a prognosis: detecting a targetentity; identifying a target entity; detecting an increase in a targetentity; detecting a decrease in a target entity.

By “progressing” or “progression” and derivations of these terms ismeant that the disease gets worse, i.e. that the severity increases. Forexample, in the case of cancer, it may mean that the tumour burdenincreases, for example a tumour increases in size and/or weight; thatthe cancer becomes malignant or more malignant; and/or that metastasisdevelops or the incidence and/or rate of metastasis increases.

The method according to various embodiments described herein mayoptionally be used to monitor the progress of disease.

During therapy or subsequent to therapy, the method of the variousembodiments described herein may optionally be used to monitor theprogress of disease to assess the effectiveness of therapy, or tomonitor the progress of therapy.

Optionally, serial (periodic) analysis of a target for a change may beused to assess whether or not therapy has been effective; the extent towhich therapy has been effective; whether or not a disease isre-occurring or progressing in the subject; and/or to assess the likelyclinical outcome (prognosis) of the disease, should it re-occur orprogress.

Optionally, the method may be used in the active monitoring of subjectswhich have not been subjected to therapy, e.g. to monitor the progressof the disease in untreated subjects. Optionally, serial (periodic)analysis of a target for a change may be used to assess whether or not,or the extent to which, the disease is progressing, thus, for example,allowing a more reasoned decision to be made as to whether therapeuticintervention is necessary or advisable.

Such monitoring may optionally be carried out on a healthy individual,e.g., an individual who is thought to be at risk of developing aparticular disease, in order to obtain an early and ideally pre-clinicalindication of the disease. Particular examples are cervical smeartesting to analyse the cervix for cancer or pre-cancerous biomarkers;and/or the analysis of the vaginal mucosa to assess the risk ofpregnancy complications such as preterm delivery.

Analytes/Biomarkers

The method according to various embodiments may involve the analysis ofanalytes, which may optionally be biomarkers. Any reference to an“analyte” should therefore be understood to encompass the embodimentthat the analyte may be a biomarker. A biomarker may be an objective,quantifiable characteristic of, e.g., a cell type, disease status,microbe, compound, and/or biological process.

By “characteristic of a cell type” is meant that the biomarker mayoptionally be used to analyse, e.g., detect, identify and/orcharacterise the cell type. Optionally, the biomarker may be used todistinguish between cells originating from different tissues; betweengenotypically and/or phenotypically different cell types; between ananimal cell and a microbial cell; between a normal and an abnormal cell;between a wild-type and a mutant cell; and/or between a diseased and ahealthy cell.

By “characteristic of a disease status” is meant that the biomarker mayoptionally be used to analyse the disease status of a target.Optionally, the biomarker may be used to distinguish between healthy anddiseased cells; and/or to analyse the severity, grade, and/or stage of adisease.

By “characteristic of a microbe” is meant that the biomarker mayoptionally be used to analyse, e.g., detect, identify and/orcharacterise the microbe. As discussed elsewhere herein, identificationmay be on any level, for example, on a taxonomic level. A biomarker thatallows identification of a microbe as belonging to a particulartaxonomic level may be referred to as a “taxonomic marker” or “taxonomicbiomarker”. Thus, a taxonomic marker may be specific for a Kingdom,Phylum, Class, Order, Family, Genus, Species and/or Strain.

By “characteristic of a compound” is meant that the biomarker mayoptionally be used to analyse, e.g., detect, identify and/orcharacterise the compound.

By “characteristic of a biological process” is meant that the biomarkermay optionally be used to analyse a biological process. Optionally, thebiomarker may be used to analyse the start, progression, speed,efficiency, specificity and/or end of a biological process.

Different cell types, disease states, compounds, microbes, biologicalprogresses and the like may be characterised by the presence or absence,and/or relative abundance, of one or more compounds, which may serve asbiomarkers. Any reference herein to a biomarker being a particularcompound, or class of compounds, should be understood optionally to bethe mass spectral data of that compound, or class of compounds.

For example, a reference to a “C24:1 sulfatide (C₄₈H₉₁NO₁₁S)” biomarkershould be understood to be a reference to the mass spectral datacorresponding to C24:1 sulfatide (C₄₈H₉₁NO₁₁S) which may, e.g., be asignal at m/z of about 888.6; whereas a reference to a “glycosylatedceramide” biomarker should be understood to be a reference to the massspectral data corresponding to glycosylated ceramide, which may, e.g.,be a signal at m/z of 842, 844 or 846.

As explained above, a biomarker may be indicative of a cell type,disease status, microbe, compound, and/or biological process. Abiomarker which is indicative of cancer may therefore be referred to asa “cancer biomarker”; a biomarker which is indicative of Pseudomonasaeruginosa may be referred to as a “Pseudomonas aeruginosa biomarker”and so on.

Optionally, a mass spectral biomarker may be identified as being themass spectral data of a particular compound, or class of compounds.Thus, a signal at a particular m/z may optionally be identified as beingindicative of the presence of a particular compound, or class ofcompounds. This may optionally involve a step of MS-MS analysis.

Optionally, mass spectral signal may serve as a biomarker even if adetermination has not been made as to which particular compound, orclass of compounds gave rise to that signal. Optionally, a pattern ofmass spectral signals may serve as a biomarker even if a determinationhas not been made as to which particular compounds, or class ofcompounds, gave rise to one or more signals in that pattern, or any ofthe signals in a pattern.

The work disclosed herein has led to the identification of a range ofbiomarkers, as well as allowing the identification of furtherbiomarkers. Optionally, the biomarker may be selected from any of thebiomarkers disclosed herein.

Optionally, the biomarker may be a biomarker of a lipid; a protein; acarbohydrate; a DNA molecule ; an RNA molecule; a polypeptide, such as,a ribosomal peptide or a non- ribosomal peptide; an oligopeptide; alipoprotein; a lipopeptide; an amino acid; and/or a chemical compound,optionally an organic chemical molecule or an inorganic chemicalmolecule.

A biomarker may optionally be the clear-cut presence or absence of aparticular compound, which may optionally manifest itself as thepresence or absence of a mass spectral signal at a specific m/z.

A biomarker may optionally be the relative abundance of a particularbiomolecule or compound, which may optionally manifest itself as therelative intensity of a mass spectral signal at a specific m/z.

A biomarker may optionally be the relative abundance of more or morecompounds, which may optionally manifest itself as the relativeintensity of two or more mass spectral signals at two or more m/z.

Thus, a biomarker may optionally be an increased or decreased level ofone or more compounds, e.g., a metabolite, a lipopeptide and/or lipidspecies, which may optionally manifest itself as an increase and/ordecrease in the intensity of two or more mass spectral signals at two ormore m/z.

The presence, absence and relative abundance of a variety of compoundsmay be referred to as a molecular “fingerprint” or “profile”. Thetotality of the lipids of a cell may be referred to as a lipidomicfingerprint/profile, whereas the totality of metabolites produced by acell may be referred to as a metabolomic fingerprint/profile.

Thus, the biomarker may be a molecular fingerprint, e.g., a lipidfingerprint and/or a metabolomic fingerprint, more particularly e.g., a(i) a lipidomic profile; (ii) a fatty acid profile; (iii) a phospholipidprofile; (iv) a phosphatidic acid (PA) profile; (v) aphosphatidylethanolamine (PE) profile; (vi) a phosphatidylglycerol (PG)profile; (vii) a phosphatidylserines (PS) profile; (viii) aphosphatidylinositol (PI) profile; or (ix) a triglyceride (TG) profile.

A lipid biomarker may optionally be selected from, e.g., fatty acids,glycerolipids, sterol lipids, sphingolipids, prenol lipids,saccharolipids and/or phospholipids.

By “metabolome” is meant a collection of the metabolites produced by acell. The metabolome may be the entirety of the cell's metabolites, or aparticular subset thereof, e.g. the subset of the 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 most abundantmetabolites produced by that cell. Metabolomics is the study of themetabolome. A metabolomic marker is a marker of one or more metabolites,or of the metabolome.

Analysis of Compounds

The method according to various embodiments may optionally involve theanalysis of one or more compounds that may be present in the biologicalsample, e.g. the mucosa, the biopsy, the body fluid and/or the faecalspecimen. Thus, the method may involve the analysis of the presence orabsence, and/or the relative abundance and/or distribution, of one ormore compounds.

Unless otherwise stated, the terms “compound”, “molecule”, “substance”and “biomolecule” are used interchangeably herein.

The compound may optionally be intracellular and/or extracellular. Itmay optionally be endogenous, i.e. produced by the subject or microbe,and/or exogenous, i.e. added to the subject, tissue, cell, and/ ormicrobe.

The compound may optionally comprise or consist of any of the compoundsor classes of compounds mentioned herein, e.g. any of the biomarkersmentioned herein. Optionally, it may comprise or consist of, forexample, a lipid, such as, a glycolipid or phospholipid; carbohydrate;DNA; RNA; protein; polypeptide, such as, a ribosomal peptide or anon-ribosomal peptide; oligopeptide; lipoprotein; lipopeptide; aminoacid; and/or chemical molecule, optionally an organic chemical molecule.

The compound may optionally be linear, cyclic or branched.

The compound may optionally be a metabolite, such as, a primary or asecondary metabolite; an antibiotic; a quorum sensing molecule; a fattyacid synthase product; a pheromone; and/or a biopolymer.

The compound may optionally be characterised by one or more of thefollowing functional groups: alcohol, ester, alkane, alkene, alkyne,ether, ketone, aldehyde, anhydride, amine, amide, nitrile, aromatic,carboxylic acid, alkyl halide, and/or carbonyl. Optionally, it mayadditionally be identified as being primary, secondary or tertiary,e.g., a primary alcohol, a secondary amine, or the like.

Optionally, the compound may be a therapeutic drug, an illicit drug, adoping agent, and/or a metabolite or derivative of any thereof.

It may optionally be selected, e.g., from any of the drugs or agentsmentioned herein, and/or Mescaline, PCP (Phencyclidine), Psilocybin,LSD, Heroin, Morphine, Codeine, dextroamphetamine, bupropion, cathinone,lisdexamfetamine, Allobarbital, Alphenal (5-allyl-5-phenylbarbituricacid), Amobarbital, Aprobarbital, Brallobarbital, Butobarbital,Butalbital, Cyclobarbital, Methylphenobarbital, Mephobarbital,Methohexital, Pentobarbital, Phenobarbital, Secobarbital, Talbutal,Thiamylal, and/or Thiopental. Ranitidine, phenylalanine PKU,dimethylamylamine , cocaine, diazepam, androstadienedione,stigmastadienone, androsteronehemisuccinate,5α-androstan-3β,17β-diol-16-one, androsterone glucuronide,epitestosterone, 6-dehydrocholestenone, phenylalanine, leucine, valine,tyrosine, methionine, sitamaquine, terfenadine, prazosin, methadone,amitripyline, nortriptyline, pethidine, DOPA, ephedrine, ibuprofen,propranolol, atenolol , acetaminophen, bezethonium, citalopram,dextrorphan, paclitaxel, proguanil, simvastatin, sunitinib, telmisartan,verapamil, amitriptyline , pazopanib, tamoxifen, imatinib,cyclophosphamide, irinotecan, docetaxel, topotecan , acylcarnitines(C2-C18), nicotine, cotinine, trans-3′-hydroxycotinine, anabasine,amphetamine, amphetamine-like stimulants, methamphetamine, MDA, MDMA,MDEA, morphine, Δ⁹-THC, tacrolimus , benzethonium , meprobamate,O-desmethyl-cis-tramadol, carisoprodol, tramadol, nordiazepam, EDDP,norhydrocodone, hydromorphone, codeine, temazepam, noroxycodone,alprazolam, oxycodone, buprenorphine, norbuprenorphine, fentanyl,propoxyphene, 6-monoacetylmorphine , caffeine, carbadox, carbamazepine,digoxigenin, diltiazem, diphenhydramine, propanolol, sulfadiazine,sulfamethazine, sulfathiazole, thiabendazole , ketamine, norketamine,BZE, AMP, MAMP, and/or 6-MAM.

Although the present invention has been described with reference topreferred embodiments, it will be understood by those skilled in the artthat various changes in form and detail may be made without departingfrom the scope of the invention as set forth in the accompanying claims.

1. A mass and/or ion mobility spectrometer comprising: a first devicearranged and adapted to accommodate a biopsy sample; a second devicearranged and adapted to generate analyte ions from a biopsy samplewithin said first device, wherein said second device is arranged andadapted to generate first analyte ions from a first position on saidbiopsy sample at a first time, and to generate second analyte ions froma second different position on said biopsy sample at a second differenttime; and an analyser arranged and adapted to analyse said analyte ions.2. The mass and/or ion mobility spectrometer as claimed in claim 1,wherein said biopsy sample comprises a sample of tissue having alongitudinal length, wherein the composition of said sample of tissuevaries or changes along said longitudinal length, and wherein saidlongitudinal length corresponds to the depth within a tissue.
 3. Themass and/or ion mobility spectrometer as claimed in claim 1, whereinsaid biopsy sample comprises a biopsy core or cylinder, and wherein saidfirst device comprises a channel arranged and adapted to accommodate abiopsy core.
 4. The mass and/or ion mobility spectrometer as claimed inclaim 1, wherein said second device is arranged and adapted to generatefirst analyte ions from a first position along the longitudinal lengthof said biopsy sample at said first time, and to generate second analyteions from a second different position along the longitudinal length ofsaid biopsy sample at said second different time.
 5. The mass and/or ionmobility spectrometer as claimed in claim 1, wherein said second deviceis arranged and adapted to scan at least a portion of the longitudinallength of said biopsy sample so as to generate analyte ions frommultiple positions along the longitudinal length of said biopsy sample.6. The mass and/or ion mobility spectrometer as claimed in claim 1,wherein said second device comprises an ambient ionisation ion source.7. The mass and/or ion mobility spectrometer as claimed in claim 6,wherein said ambient ionisation ion source comprises an ion sourceselected from the group consisting of: (i) a rapid evaporativeionisation mass spectrometry (“REIMS”) ion source; (ii) a desorptionelectrospray ionisation (“DESI”) ion source; (iii) a laser desorptionionisation (“LDI”) ion source; (iv) a thermal desorption ion source; (v)a laser diode thermal desorption (“LDTD”) ion source; (vi) a desorptionelectro-flow focusing (“DEFFI”) ion source; (vii) a dielectric barrierdischarge (“DBD”) plasma ion source; (viii) an Atmospheric SolidsAnalysis Probe (“ASAP”) ion source; (ix) an ultrasonic assisted sprayionisation ion source; (x) an easy ambient sonic-spray ionisation(“EAST”) ion source; (xi) a desorption atmospheric pressurephotoionisation (“DAPPI”) ion source; (xii) a paperspray (“PS”) ionsource; (xiii) a jet desorption ionisation (“JeDI”) ion source; (xiv) atouch spray (“TS”) ion source; (xv) a nano-DESI ion source; (xvi) alaser ablation electrospray (“LAESI”) ion source; (xvii) a directanalysis in real time (“DART”) ion source; (xviii) a probe electrosprayionisation (“PESI”) ion source; (xix) a solid-probe assistedelectrospray ionisation (“SPA-ESI”) ion source; (xx) a cavitronultrasonic surgical aspirator (“CUSA”) device; (xxi) a focussed orunfocussed ultrasonic ablation device; (xxii) a microwave resonancedevice; and (xxiii) a pulsed plasma RF dissection device.
 8. The massand/or ion mobility spectrometer as claimed in claim 1, wherein saidsecond device is arranged and adapted to generate aerosol, smoke orvapour from said biopsy sample, and to ionise said aerosol, smoke orvapour in order to generate said analyte ions.
 9. The mass and/or ionmobility spectrometer as claimed in claim 1, wherein said second devicecomprises: (i) one or more electrodes arranged and adapted to contactsaid biopsy sample to generate said aerosol, smoke or vapour; and/or(ii) a laser for irradiating said sample.
 10. The mass and/or ionmobility spectrometer as claimed in claim 1, wherein said second deviceis arranged and adapted to direct ultrasonic energy into said sample.11. The mass and/or ion mobility spectrometer as claimed in claim 1,wherein said second device is arranged and adapted to direct a spray ofcharged droplets onto said biopsy sample so as to generate said analyteions.
 12. The mass and/or ion mobility spectrometer as claimed in claim11, wherein said ambient ionisation ion source comprises a desorptionelectrospray ionisation (“DESI”) ion source or a desorption electroflowfocusing ionisation (“DEFFI”) ion source, and wherein said desorptionelectrospray ionisation (“DESI”) ion source or desorption electroflowfocusing ionisation (“DEFFI”) ion source is arranged and adapted toperform a gradient desorption electrospray ionisation analysis along thelength of said biopsy sample, wherein the composition of a solventsupplied to and/or emitted from said desorption electrospray ionisation(“DESI”) ion source or desorption electroflow focusing ionisation(“DEFFI”) ion source is varied as a function of position along thelength of the biopsy sample.
 13. The mass and/or ion mobilityspectrometer as claimed in claim 1, wherein said analyser comprises: (i)a mass analyser or filter and/or ion mobility spectrometer for massanalysing and/or ion mobility analysing said analyte ions and/or ionsderived from said analyte ions; (ii) an ion mobility device fordetermining the ion mobility, collision cross section or interactioncross section of said analyte ions and/or ions derived from said analyteions; and/or (iii) one or more fragmentation, collision or reactiondevices for fragmenting or reacting said analyte ions.
 14. A method ofmass spectrometry and/or method of ion mobility spectrometry comprising:providing a biopsy sample; generating first analyte ions from a firstposition on said biopsy sample at a first time, and generating secondanalyte ions from a second different position on said biopsy sample at asecond different time; and analysing said analyte ions.
 15. A methodcomprising: sampling tissue using a biopsy needle so as to produce afirst biopsy sample and a second biopsy sample; analysing said firstbiopsy sample in a first mode of operation, wherein said first mode ofoperation comprises generating analyte ions from said first biopsysample and analysing said analyte ions; and analysing said second biopsysample in a second different mode of operation.
 16. The method asclaimed in claim 15, wherein said first mode of operation comprisesgenerating analyte ions from said first biopsy sample using a firstambient ionisation analysis method, and wherein said second mode ofoperation comprises generating analyte ions from said second biopsysample and analysing said analyte ions in a second different mode ofoperation.
 17. The method as claimed in claim 16, wherein said secondmode of operation comprises generating analyte ions from said secondbiopsy sample using said first ambient ionisation analysis method in asecond different mode of operation.
 18. The method as claimed in claim16, wherein said second mode of operation comprises generating aplurality of analyte ions from said second biopsy sample using a seconddifferent ambient ionisation analysis method.
 19. The method as claimedin claim 15, wherein said second different mode of operation comprises:(i) a gene sequencing mode of operation; (ii) a Matrix-Assisted LaserDesorption Ionisation (“MALDI”) mode of operation; and/or (iii) ahistopathological mode of operation.
 20. The method as claimed in claim15, wherein: said first biopsy sample comprises a first portion oftissue and said second biopsy sample comprises a second portion of saidtissue; and said first portion of said tissue was adjacent and/orconnected to said second portion of said tissue.